TECHNISCHE UNIVERSITÄT MÜNCHEN

Lehrstuhl für Humanmedizin

Positive allosteric modulation of GABAA

receptors in the amygdala, hippocampus and

thalamocortical circuits by XBD173, diazepam

and allopregnanolone: a comparative VSDI

study

Tatiana Engel

2017

TECHNISCHE UNIVERSITÄT MÜNCHEN

Fakultät für Medizin

Positive allosteric modulation of GABAA receptors

in the amygdala, hippocampus and thalamocortical circuits by

XBD173, diazepam and allopregnanolone: a

comparative VSDI study

Tatiana N. Engel

Vollständiger Abdruck der von der Fakultät für Medizin der Technischen Universität

München zur Erlangung des akademischen Grades eines

Doktors der Medizin

genehmigten Dissertation.

Vorsitzender: Prof. Dr. Ernst J. Rummeny

Prüfer der Dissertation: 1. apl. Prof. Dr. Gerhard Rammes

2. Prof. Dr. Johann Förstl

Die Dissertation wurde am 25.10.2017 bei der Technischen Universität München

eingereicht und durch die Fakultät für Medizin am 10.10.2018 angenommen.

Meinen Eltern gewidmet

IV

Table of contents:

Table of figures ............................................................................................................................... VII

Table of Abbreviations ..................................................................................................................... IX

1 Introduction ................................................................................................................................. 1

1.1 Anxiety Disorders ................................................................................................................ 1

1.2 Pharmacological Treatment of Anxiety Disorders .............................................................. 1

1.3 Benzodiazepines ................................................................................................................ 2

1.4 Neurosteroids...................................................................................................................... 2

1.5 Translocator protein 18 kDa ............................................................................................... 4

1.5.1 Location and structure ................................................................................................. 5

1.5.2 TSPO function ............................................................................................................. 5

1.5.3 Ligands to TSPO ......................................................................................................... 7

1.5.4 XBD173 ....................................................................................................................... 7

1.6 GABAA receptor .................................................................................................................. 8

1.7 Amygdala .......................................................................................................................... 10

1.7.1 Anatomy and Neurophysiology ................................................................................. 10

1.7.2 Function ..................................................................................................................... 10

1.8 Hippocampus .................................................................................................................... 11

1.8.1 Neurophysiology ........................................................................................................ 11

1.8.2 Function ..................................................................................................................... 12

1.9 Thalamocortical Connections ........................................................................................... 12

1.9.1 Anatomy and Neurophysiology ................................................................................. 12

1.9.2 Function ..................................................................................................................... 14

1.10 Aim of this dissertation ...................................................................................................... 14

V

2 Material and Methods ............................................................................................................... 16

2.1 Preparation of brain slices ................................................................................................ 16

2.2 Voltage sensitive dye imaging .......................................................................................... 17

2.2.1 Staining of brain slices with Di-4-ANEPPS ............................................................... 17

2.2.2 VSDI Setup ................................................................................................................ 19

2.2.3 Recordings ................................................................................................................ 20

2.3 Data analysis .................................................................................................................... 22

3 Results ..................................................................................................................................... 24

3.1 Alteration of neuronal activity upon stimulus in the Amygdala by diazepam, XBD and

allopregnanolone ........................................................................................................................ 24

3.1.1 Effect of diazepam on neuronal activity upon stimulus in the amygdala .................. 24

3.1.2 Effect of XBD on neuronal activity upon stimulus in the amygdala .......................... 25

3.1.3 Effect of allopregnanolone on neuronal activity upon stimulus in the amygdala ...... 27

3.1.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in the

amygdala ................................................................................................................................ 28

3.2 Alternation of neuronal activity upon stimulus in the hippocampus by diazepam, XBD and

allopregnanolone ........................................................................................................................ 28

3.2.1 Effect of diazepam on neuronal activity upon stimulus in the hippocampus ............ 28

3.2.2 Effect of XBD on neuronal activity upon stimulus in the hippocampus .................... 30

3.2.3 Effect of allopregnanolone on neuronal activity upon stimulus in the hippocampus 31

3.2.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in the

hippocampus .......................................................................................................................... 32

3.3 Alternation on neuronal activity upon stimulus in the thalamocortical connections by

diazepam, XBD and allopregnanolone ...................................................................................... 33

3.3.1 Effect of diazepam on neuronal activity upon stimulus in the thalamocortical

connections ............................................................................................................................ 33

VI

3.3.2 Effect of XBD on neuronal activity upon stimulus in the thalamocortical connections

35

3.3.3 Effect of allopregnanolone on neuronal activity upon stimulus in the thalamocortical

connections ............................................................................................................................ 37

3.3.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in the

thalamocortical connections ................................................................................................... 38

3.3.5 Analyses of diazepam, XBD and allopregnanolone impact on the individual layers of

the cortex ................................................................................................................................ 40

4 Discussion ................................................................................................................................ 42

4.1 Result interpretation .......................................................................................................... 42

4.1.1 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the amygdala

42

4.1.2 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the

hippocampus .......................................................................................................................... 43

4.1.3 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the

thalamocortical connections ................................................................................................... 43

4.2 Clinical relevance, sources of error and outlook .............................................................. 45

4.3 Conclusion ........................................................................................................................ 45

5 Abstract .................................................................................................................................... 47

6 References ............................................................................................................................... 48

7 Appendix .................................................................................................................................. 60

Used Substances ........................................................................................................................... 60

Acknowledgements ........................................................................................................................ 62

VII

Table of figures

Fig. 1: Synthesis of neurosteroids and schematic sketch of allopregnanolone effect ..................... 3

Fig. 2: TSPO relevance in anxiolysis ............................................................................................... 6

Fig. 3: Scheme of XBD’s mode of action and display of GABAA receptor ....................................... 9

Fig. 4: Schematic display of hippocampal formation and the trisynaptic circuit ............................ 11

Fig. 5: Schematic sketch of the thalamocortical connections ........................................................ 13

Fig. 6: Scheme of brain regions associated with benzodiazepine effects and adverse effects .... 15

Fig. 7: Schematic sketch of the principle of voltage sensitive shift of emission ............................ 18

Fig. 8: Schematic sketch of the VSDI-setup and presentation of the processed signal ................ 20

Fig. 9: Display of the slice preparations and electrode and ROI placement .................................. 21

Fig. 10 Excitation transmission in the thalamocortical circuit ........................................................ 24

Fig. 11: Diazepam impact on neuronal activity in the amygdala ................................................... 25

Fig. 12: XBD impact on neuronal activity in the amygdala ............................................................ 26

Fig. 13: allopregnanolone impact on neuronal activity in the amygdala ........................................ 27

Fig. 14 Comparison of substance effects on FDSAUC, FDSmaxInt and area in the amygdala ......... 28

Fig. 15: diazepam impact on neuronal activity in the hippocampus .............................................. 29

Fig. 16: XBD impact on neuronal activity in the hippocampus ...................................................... 30

Fig. 17: allopregnanolone impact on neuronal activity in the hippocampus .................................. 31

Fig. 18 Comparison of substance effects on FDSAUC, FDSmaxInt and area in the hippocampus ... 32

Fig. 19: diazepam impact on neuronal activity in the thalamocortical connections ....................... 34

Fig. 20: XBD impact on neuronal activity upon stimulus in the thalamocortical circuit.................. 36

Fig. 21: allopregnanolone impact on neuronal activity upon stimulus in the thalamocortical

connections ............................................................................................................................. 38

Fig. 22 Comparison of substance effects on FDSAUC FDSmaxInt and area in the thalamocortical

connections ............................................................................................................................. 39

VIII

Fig. 23 Display of cortex division for layer analysis ....................................................................... 40

Fig. 24 Layer analysis of diazepam and XBD impact on normalized maximal Intensity ............... 41

IX

Table of Abbreviations

3,5-THDOC allotetrahydrodeoxycorticosterone, 3,21-Dihydroxy-5-pregnan-20-one

a area [No of pixel]

ACSF artificial cerebrospinal fluid

Allo allopregnanolone, 5-pregnan-3-ol-20-one, 3-5-THP

ANT adenine nucleotide translocase

CCD charged-coupled device

CCK4 cholecystokinin tetrapeptide

Cl- Chloride Ion

CNS central nervous system

Diaz diazepam

DMSO dimethyl sulfoxide

eEPSC electric evoked excitatory postsynaptic current

EPSC excitatory postsynaptic current

F/XBD finasteride-Experiments

FBS fetal bovine serum

FDS fast depolarization signal

Fina finasteride

Fluma flumazenil

GABA γ-Aminobutyric acid

GABAA-R GABAA-Receptor

HR-CCD high-resolution charge-coupled device

Int Intensity [∆F/F]

Intmax maximal change of Intensity (layer analysis)

X

IPSC inhibitorischer postsynaptic current

n number of experiments performed

P450scc Cholesterol side chain cleavage cytochrome P450

PAM positive allosteric modulator

PK 11195 N-butan-2-yl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide, TSPO Antagonist

PSC Postsynaptic current

RAS reticular activating system

ROI region of interest

SEM standard error of the mean

SSRI selective serotonin reuptake inhibitor

TC thalamocortical neuron, also named relay neuron

TRN Thalamic reticular neuron

TSPO Translocator Protein (18 kDa (kilo Dalton)), formerly called: peripheral benzodiazepine receptor

VDAC voltage-dependent anion channel

XBD XBD-173, also named Emapunil or AC-5216, systematic name: N-benzyl-N-ethyl-2(7-methyl-8-oxo-2-phenylpurin-9-yl)acetamide

Introduction Anxiety Disorders

1

1 Introduction

1.1 Anxiety Disorders

Anxiety disorders are highly prevalent in today’s society. They are amongst the most common

psychiatric illnesses. Prevalence and lifetime risk differ considerably between countries. In

Germany the projected lifetime risk is about 17%, whereas in the USA it is as high as 36% (Kessler

et al. 2007). Anxiety disorders comprise a number of distinct diagnoses. They can further be

categorized into panic disorders, specific phobias, social phobias, post-traumatic stress disorders,

obsessive-compulsive disorders and generalized anxiety disorders amongst others (Baldwin et al.

2014). A multifactorial pathogenesis is assumed, including external factors such as traumatic or

straining experiences as well as internal factors like genetics and neurobiological dysfunction

(Höffler et al. 2003). While symptoms can be minor and temporarily, a lot of patients suffer from

severe and recurring symptoms that impair their quality of life to an extent that requires treatment.

1.2 Pharmacological Treatment of Anxiety Disorders

Therapeutic approaches include psychotherapies and psychopharmacological drugs (Baldwin et

al. 2014). However, current pharmacological treatment options are limited by pharmacodynamics

and undesirable side effects. First line in treatment antidepressants such as selective serotonin

reuptake inhibitors (SSRI) exert their anxiolytic effect only after several weeks of treatment

(Bandelow et al. 2008). Though in general well-tolerated, potential initial adverse effects like

nausea, jitteriness, insomnia and even increase of anxiety symptoms as well as weight gain and

sexual dysfunction that may occur in long-term treatment are known to impair patient compliance

(Bandelow et al. 2008). More rapidly onset drugs commonly used are benzodiazepines.

Unfortunately they are not eligible either, due to their broad range of unfavorable adverse effects,

development of tolerance and danger of addiction and withdrawal symptoms.

A new class of substances with a preferable pharmacological profile is needed. However, more

suitable medication needs yet to be established. Drugs, which dispose of rapidly on setting

anxiolytic effects like benzodiazepines, but lack a broad side effect profile, are much needed.

Satisfying anxiolytic effects of endogenous neurosteroids such as allopregnanolone have been

shown (Bitran et al. 1991, Wieland et al. 1991). Unfortunately, pharmacokinetic obstacles such as

low bioavailability limit their medical use (Reddy 2010). However, there is a way to enhance natural

neurosteroid synthesis: The Translocator Protein 18 kDa (TSPO) and its ligands provide a

promising new option. TSPO is crucially involved in the synthesis of neurosteroids, for it is in charge

Introduction Benzodiazepines

2

of the rate-determining step of neurosteroidogenesis (Papadopoulos et al. 1997a). Therefore

ligands to TSPO, like XBD-173 (XBD; Emapunil), that enhance TSPO’s effects and subsequently

promote neurosteroid synthesis are currently under investigation as a potential new therapeutic

alternative, that has already revealed great anxiolytic capabilities and a favorable side effect profile

(Rupprecht et al. 2009).

1.3 Benzodiazepines

Besides in anxiety disorders Benzodiazepines are used in treatment of sleeping disorders, epilepsy

and schizophrenia (Rote-Liste 2013, 2015). They are also commonly used as premedication before

surgical interventions and for calming effects in emergencies as well as to treat muscle spasms.

Even though they are very potent anxiolytic drugs and exhibit a quick onset of anxiolytic effects

they are not suitable for long-term anxiety treatment due to a broad side effect profile, development

of tolerance and danger of addiction and withdrawal symptoms (Bandelow et al. 2008) . Side effects

include sedation, drowsiness, anterograde amnesia, respiratory depression and central diminution

of tonicity (Rote-Liste 2013, 2015).

Benzodiazepines exert their effects by enhancing γ-Aminobutyric acid (GABA) impact in the central

nervous system (CNS) (Rote-Liste 2013, 2015). Since GABA is the most important inhibitory

transmitter in the CNS, they reduce neuronal activity (Bloom et al. 1971). Benzodiazepines bind to

a specific compound of the GABAA-receptor (GABAA-R) between the α- and -subunit (Möhler

2006). Once connected to the receptor, they exert an allosteric effect that facilitates binding of

GABA to the GABAA-R and increases its opening frequency (see chapter 1.6), thus decreasing

neuronal excitability, which causes anxiolytic effects amongst other things (Walters et al. 2000).

1.4 Neurosteroids

Neuroactive steroids are a specific kind of steroids that, in addition to genomic alternation, the

common course of action of steroids, express the ability to modulate neurotransmitter receptors

(Majewska et al. 1986, Paul et al. 1992, Rupprecht et al. 1999). While gene expression via steroid

activated transcriptional factors take minutes to hours, effects by modulation of neurotransmitter

receptors occur within seconds (McEwen 1991). One of the affected receptors is the GABAA-R

(Majewska et al. 1986).

Neurosteroids are predominantly produced in the brain and independent of peripheral steroid levels

(Corpéchot et al. 1981). The precursor of all neurosteroids is pregnenolone, which is synthesized

out of cholesterol inside the mitochondrion (Lacapère et al. 2003, Rupprecht et al. 2010). Synthesis

of pregnenolone underlies regulation by TSPO (see chapter 1.5.2). Pregnenolone leaves the

Introduction Neurosteroids

3

mitochondrion and is processed further in the cytosol into a variety of other neurosteroids (see Fig.

1A).

A

B

Fig. 1: Synthesis of neurosteroids and schematic sketch of allopregnanolone effect

A Scheme of synthesis of neurosteroids from (Schüle et al. 2014) according to (Nothdurfter et al. 2012a). Neurosteroids are formed out of cholesterol. First step, transformation to pregnenolone, the precursor of all neurosteroids, takes place in the mitochondrion. Further synthesis of diverse neurosteroids out of pregnenolone takes place in the cytoplasm. Utilized enzymes are depicted as arrows. B Scheme of allopregnanolone effect according to (Nothdurfter et al. 2012b). Allopregnanolone binds to the GABAA receptor where it exerts a modulating effect, enhancing GABA binding and subsequent chloride influx, thus potentiating its inhibitory impact on neuronal activity, which leads to Anxiolysis.

Introduction Translocator protein 18 kDa

4

Neurosteroid biosynthesis is region and neuron specific, depending on the expression of the

respective enzymes for neurosteroid formation (Rupprecht et al. 2010). Region-specific synthesis

of neurosteroids is subsequently responsible for a distinct modulation of neurotransmitter receptors

in certain brain areas, resulting in region-specific alteration of signal transduction. Some of the

neurosteroids formed are 3-reduced neurosteroids, such as allopregnanolone (Rupprecht 1997).

Certain 3-reduced steroids, especially allopregnanolone, have been shown to feature anxiolytic

properties in various stress-models in rodents (Bitran et al. 1991, Picazo et al. 1995, Wieland et al.

1995, Reddy et al. 1997). Furthermore they may play a role in pathology of affective diseases

including anxiety disorders (Strohle et al. 2003, Reddy 2010). Similar to benzodiazepines, 3-

reduced steroids exercise their anxiolytic effects by potentiating inhibitory effects of GABA (see

Fig. 1B) (Gee 1988, Bitran et al. 1991). However they modulate GABAA-receptors using another

binding site (see Fig. 3B) (Reddy et al. 1997, Hosie et al. 2006).

Allopregnanolone stands out with particular effectiveness in reducing stress and anxiety-like

behavior in rodents. Increased allopregnanolone levels were detected in rodents (also

adrenalectomized rodents) exposed to stressors or acute stress paradigms (Purdy et al. 1991,

Barbaccia et al. 1997). Induced panic attacks in patients with panic disorders led to decreasing of

allopregnanolone plasma concentrations (Strohle et al. 2003). While stressors raise

allopregnanolone levels, presumably attempting to prevent anxiety or panic outbreak, panic attacks

reduce allopregnanolone levels assumedly by consumption. Anti-anxiety behavior in rodents has

been reduced by blockage of allopregnanolone formation (Frye et al. 2011, Koonce et al. 2012).

Concordantly, anti-anxiety behavior was reinstated by allopregnanolone injection (Frye et al. 2011).

These findings propose allopregnanolone to be a neurosteroid of particular importance concerning

anxiety treatment (Schüle et al. 2014).

Unfortunately natural neurosteroids like allopregnanolone have a low bioavailability as a result of

rapid inactivation and elimination by glucuronidation and sulfate conjugation, as well as possible

oxidation to ketone (Reddy 2010). Hence, they are not viable for therapeutic use. Fortunately there

are methods to enhance neurosteroid synthesis in the brain as described in the following chapters.

1.5 Translocator protein 18 kDa

The Translocator protein (18 kDa) (TSPO) was first recognized in 1977 in the kidney on the search

for binding sites for benzodiazepines (Braestrup et al. 1977) and thus afterwards named peripheral

benzodiazepine receptor. In consideration of its broad range of function, employment beyond its

receptor capacities and due to the fact that not all benzodiazepines bind to it, but a number of other

substances do and that its structure and function differ tremendously from the central

Introduction Translocator protein 18 kDa

5

benzodiazepine receptor (part of the GABAA receptor complex), it was renamed Translocator

protein (18 kDa) in 2006 (Papadopoulos et al. 2006a).

1.5.1 Location and structure

TSPO has since been detected in various tissues including the brain, though the highest levels of

expression have been found in steroid producing tissues like adrenal and gonad (Anholt et al. 1985,

Gavish et al. 1999). Inside the CNS it is primarily expressed in glia cells (Gavish et al. 1999).

At the subcellular level, TSPO is mainly – though not exceptionally (Olsen et al. 1988, Oke et al.

1992) – located at the outer mitochondrial membrane (Anholt et al. 1986), cumulating at contact

sites of the outer and inner mitochondrial membrane (Culty et al. 1999). Topological analysis of

TSPO revealed it to consist of 5 trans membrane -helices (Joseph-Liauzun et al. 1998). 3D

modeling of TSPO suggested that the helices form a channel, likely used for cholesterol transport

(Bernassau et al. 1993, Culty et al. 1999). However to be accurate TSPO should be characterized

as a cholesterol exchanger rather than a channel, for it actively transports cholesterol instead of

just letting it pass (Lacapère et al. 2003).

TSPO interacts with other mitochondrial membrane proteins such as the 32 kDa voltage dependent

anion channel (VDAC) and the 30 kDa adenine nucleotide translocase (ANT) (McEnery et al.

1992). They form a complex reaching from outer to inner mitochondrial membrane, whereupon

TSPO is the minimal functional unit able to bind cholesterol and drug ligands (Lacapère et al. 2001).

However VDAC is essential for maximizing benzodiazepine and endozepine (endogenous

compounds with likewise effects as benzodiazepines) binding to TSPO (Garnier et al. 1994). The

entire impact of these protein associations is not yet fully understood, but they are believed to alter

drug binding properties and related functions (Golani et al. 2001). Furthermore,

homopolymerization of TSPO influences pharmacological and physiological properties (Delavoie

et al. 2003).

1.5.2 TSPO function

TSPO is expressed of a gene highly conserved throughout evolution, appearing in all sorts of

organisms extending from bacteria to humans (Gavish et al. 1999). It has been found to participate

in many cell regulation and signal transduction mechanism of the mitochondrion. Three main

functions are thought to be primarily responsible for all TSPO-delegated effects (Papadopoulos et

al. 2006a). These include cholesterol (Papadopoulos et al. 1997b, Li et al. 2001), protein (Wright

et al. 1999, Hauet et al. 2005) and porphyrin (Synder et al. 1987, Taketani et al. 1994) binding and

transport into the mitochondrion. Cholesterol translocation is essential for steroid and bile salt

biosynthesis, protein import is necessary for membrane biogenesis and porphyrin import is a part

of the heme biosynthesis pathway (Papadopoulos et al. 2006a). Associated functions might include

Introduction Translocator protein 18 kDa

6

cellular proliferation and differentiation, immunomodulation, cellular respiration and involvement in

oxidative processes, regulation of mitochondrial metabolism and apoptosis (Papadopoulos et al.

2006a, Rupprecht et al. 2010). The importance of TSPO for the organism is demonstrated by the

fetal consequence of functional deactivation in an early embryotic phase in mice (Papadopoulos et

al. 1997a).

As for TSPO involvement in neurosteroid

genesis, cholesterol translocation from the

cytosol to the inner mitochondrial membrane

by TSPO (Papadopoulos et al. 1997a) is the

rate-limiting step in neurosteroid genesis

(Jefcoate 2002). Inside the Mitochondrion is

the cholesterol side chain cleavage

cytochrome enzyme P450 (P450scc), ready to

metabolize cholesterol to pregnenolone, the

precursor of all neurosteroids (Lacapère et al.

2003, Rupprecht et al. 2010). Positive

allosteric neurosteroids such as

allopregnanolone are subsequently formed

and modulate GABAA receptors, thus exerting

anxiolytic effects (Fig. 2) (Papadopoulos et al.

2006b). Since inside the CNS, TSPO is mainly

expressed in glia cells, the acquired

neurosteroids are believed to work in a

paracrine fashion for the most part, operating

at receptors located at surrounding neurons

(Nothdurfter et al. 2012a).

In addition TSPO seems to be a considerable

factor in a number of pathological conditions,

including brain injury, peripheral neuropathy,

neurodegenerative disorders such as Alzheimer’s disease and Parkinson’s disease, epilepsy,

certain kinds of cancer and psychiatric disorders such as anxiety disorders (Papadopoulos et al.

2006a). As a matter of fact a number of studies demonstrated an increase in TSPO density in acute

stress while under chronic stress as well as in anxiety disorders density is usually decreased,

indicating TSPO to be a critical factor in the organism’s way of coping with stress (Gavish et al.

1999). For instance, in patients suffering from generalized anxiety disorder, lower levels of ligand

binding to TSPO have been measured (Weizman et al. 1987). Moreover, levels increased again

Fig. 2: TSPO relevance in anxiolysis

Illustration of TSPO function in anxiolysis according to (Nothdurfter et al. 2012b). Cholesterol is imported to the inside of the mitochondrion by TSPO. Here the first step of neurosteroidogenesis, pregnenolone (precursor of all neurosteroids) formation out of cholesterol, is performed. Subsequent, allopregnanolone is increasingly synthesized and ready to manipulate GABAA-Receptors, resulting in anxiolysis.

Introduction Translocator protein 18 kDa

7

after treatment with diazepam whereupon relief of anxiety was stated (Weizman et al. 1987). Also

in soldiers repeatedly exposed to stress exercises, reduced PBR density was monitored as well as

in civilians during war (Dar et al. 1991, Weizman et al. 1994). Whereas in psychiatric residents

completing their exams significant increase of platelet PBR density was shown, presumably as a

way to cope with stress and prevent anxiety (Karp et al. 1989). Therefore, drugs acting at a

mechanism that appears to be involved in pathology of anxiety formation propose to show great

promise.

1.5.3 Ligands to TSPO

There are a number of substances capable of binding to TSPO. Within high affinity endogenous

ligands are cholesterol and porphyrins. Also endozepines (derivatives of the common polypeptide

precursor diazepam-binding inhibitor (DBI)) are to be mentioned, which are able to displace

benzodiazepines from their binding site. While cholesterol is known to connect to the C-terminus

of TSPO (Li et al. 2001), drug ligands bind to the amino terminus (Rupprecht et al. 2010). This is

consistent with the observation that cholesterol is imported to the inner mitochondrial membrane

by TSPO, while other ligands influence TSPO properties such as cholesterol translocation

(Lacapère et al. 2003). Endogenous drugs are therefore likely to regulate TSPO function. Given

the many TSPO functions and its involvement in pathologies, great therapeutic benefit is to be

expected of synthetic TSPO ligands.

A number of synthetic ligands have been developed. A lot of them initially served as neuroimaging

agents to determine TSPO distribution or for means of identification of TSPO function (Rupprecht

et al. 2010). However certain ligands have been shown to promote neurosteroidogenesis in the

brain and therefore exercise anxiolytic effects in rodent models (Serra et al. 1999, Bitran et al.

2000, Verleye et al. 2005). One of them is XBD173 (XBD) (Rupprecht et al. 2009).

1.5.4 XBD173

XBD’s mode of action

XBD, a phenyl-purine acetamide with promising anxiolytic potential, is a selective ligand to TSPO

with nanomolar affinity (Kita et al. 2004). Its affinity to neurotransmitter receptors including the

GABAA receptor is negligible (Kita et al. 2004). A research group around Rupprecht (2009)

demonstrated that XBD does not exert any direct effects at the GABAA receptor. However,

appliance of XBD to neocortical slices potentiated GABAergic neurotransmission (Rupprecht et al.

2009). This was prevented by finasteride, a 5-reductase inhibitor, indicating 5-reduced

neurosteroids to be responsible for GABA-mediated actions following XBD application (Rupprecht

et al. 2009). Furthermore, significantly elevated levels of allopregnanolone in the brains of rats were

measured after oral medication with XBD (Rupprecht et al. 2009). In addition, anxiolytic properties

Introduction GABAA receptor

8

of XBD during stress tests (social exploration and elevated plus maze test (Pellow et al. 1985)) in

rats were prevented by the TSPO antagonist PK 11195, confirming XBD’s mode of action to be

TSPO-mediated up-regulation of neurosteroidogenesis (Fig. 3) (Rupprecht et al. 2009).

Effect and side-effect profile of XBD

Besides its anxiolytic abilities in stress tests, XBD also counteracted sodium lactate and

cholecystokinin tetrapeptide (CCK4) induced panic attacks in rats (Rupprecht et al. 2009). Hereby

no signs of sedation were detected, different to like-wise application of the benzodiazepine

alprazolam. Moreover, there was no indication for decrease of XBD’s anxiolytic efficiency

(tolerance) in the social exploration stress test under subchronic administration, a phenomenon

well known in benzodiazepine-treatment (Rupprecht et al. 2009, Rote-Liste 2013, 2015). This was

also observed in anxiety models in mice by the research team around Kita (Kita et al. 2009). In

addition, XBD unlike diazepam lacked withdrawal symptoms in special designed withdrawal

experiments they performed on mice (Kita et al. 2009).

In humans, XBD revealed anxiolytic properties as well. Anxiolytic efficiency of XBD proved to be

as potent as alprazolam’s in the CCK-4 challenge (Rupprecht et al. 2009). Occurrence of side

effects in XBD treated subjects was comparable to incidents in subjects treated with placebo.

Alprazolam treated subjects however suffered a much higher incidence of adverse effects,

especially dizziness and somnolence. The same was reported of withdrawal symptoms.

To summarize, the data presented by Rupprecht at al (2009) and Kita at al (2009) indicates that

XBD possesses rapidly onset anxiolytic properties comparable to benzodiazepines, but with a

distinct superior side effect profile. More precisely, as far as up to date studies detected, XBD lacks

adverse effects like sedation, anterograde amnesia, tolerance development, addiction and

withdrawal symptoms. Whereas all of the above have been observed in patients treated with

benzodiazepines (Rote-Liste 2013, 2015). Therefore XBD may be a superior candidate in the

treatment of anxiety disorders.

1.6 GABAA receptor

The diverse side effect profile of XBD and diazepam despite corresponding final modes of action

could be due to different distribution of the various subtypes of GABAA receptors. Though both,

benzodiazepines and certain neurosteroids (5-reduced neurosteroids such as allopregnanolone

and 3,5-THDOC), act as positive allosteric modulators at the GABAA receptor, they occupy

different binding sites (Fig. 3B), which do not always coexist in the same GABAA receptor (Olsen

et al. 2008).

Introduction GABAA receptor

9

A B

Fig. 3: Scheme of XBD’s mode of action and display of GABAA receptor

A: TSPO ligand mediated neurosteroidogenesis; modified according to (Rupprecht et al. 2010). TSPO ligands, like XBD, enhance TSPO function, thus favoring translocation of Cholesterol from the cytosol to the inner mitochondrial membrane, the rate-limiting step in neurosteroid genesis. The cholesterol side-chain-cleaving cytochrome P450 enzyme (P450scc) metabolizes cholesterol to pregnenolone (Pregnenol.), the precursor of all neuroactive steroids. In the cytosol Pregnenolone is metabolized

further. For allopregnanolone formation the enzymes 5-Reductase and 3-Hydroxysteroid

dehydrogenase (3-HSD) amongst others are necessary. Allopregnanolone then exerts anxiolytic effects by modulation of the GABAA receptor. B: systematic display of the GABAA receptor including various binding sites; according to (Nothdurfter et al. 2012a). The GABAA receptor is located at the cell membrane. It is build out of 5 subunits deriving out of 8 subunit classes. Different binding sides for GABA, diazepam (in between α- and γ-subunits) and allopregnanolone (in between α- and β- subunits) can be identified.

-Aminobutyric acid (GABA) is the main inhibitory neurotransmitter receptor in the CNS (Bloom et

al. 1971). The GABAA receptor belongs to the category of ligand-gated ion channels. It is built of 5

subunits, each containing 4 transmembrane domains, forming a central pore (Bormann et al. 1987,

Pirker et al. 2000). Upon binding of two GABA molecules, the pore becomes permeable for chloride

ions (Cl-) and bicarbonate (HCO3-) (Bormann et al. 1987, Macdonald et al. 1994). Influx of negative

charged ions induces hyperpolarization, causing an inhibitory postsynaptic current to occur in the

corresponding neuron, cancelling out potential excitatory postsynaptic currents emerging

simultaneously from another synapse of the same neuron. In short: GABA has a diminishing impact

on neuronal activity e.g. signal transduction.

Introduction Amygdala

10

The subunits forming the GABAA receptor can vary out of a range of 19 different subunits divided

in 8 subunit-classes: 1-6, 1-3, 1-3, , , , , 1-3. (Barnard et al. 1998, Olsen et al. 2008).

Depending on the subunit composition, the GABAA receptor is able to bind a variety of different

drugs and endogenous ligands. Distribution of diverse GABAA receptor subtypes in the brain is

heterogeneous (Pirker et al. 2000).

Benzodiazepines and certain neurosteroids (5-reduced neurosteroids such as allopregnanolone

and THDOC) both facilitate GABA binding by enhancing receptor affinity towards GABA mediated

by allosteric changes of the GABAA receptor (Sigel et al. 1997, Belelli et al. 2005). While the binding

site for benzodiazepines is formed by - and -subunits, neurosteroids occupy another binding site

located in between the - and - subunits (Fig. 3) (Hosie et al. 2006, Möhler 2006). Therefore, not

only availability of benzodiazepines or neurosteroids is necessary for respective effects, but also

the expression of corresponding GABAA receptor subtypes. This might be a significant factor,

leading to the disparity of their side effect profiles.

1.7 Amygdala

The main focus of this dissertation is on the discordance between the side effect profile of XBD

and diazepam. This discordance is most likely due to different effects that these drugs have on

respective parts of the brain. Hence the influence of those substances on the amygdala, the

hippocampus and the thalamocortical connections was measured in the course of this dissertation.

Those cerebral areas will therefore be introduced in this and the following chapters.

1.7.1 Anatomy and Neurophysiology

The corpus amygdaloideum, more commonly known as the amygdala, is localized in the medial

temporal lobe in each hemisphere. The amygdala consists of many distinct, however densely

interconnected, nuclei (Amaral et al. 1992). They can be functionally and anatomically grouped into

three core domains: the basolateral (composed of the lateral and the basal nuclei (Keifer et al.

2015)), the centromedial and the superficial nuclei group (Bzdok et al. 2013). The basolateral

amygdala obtains its information from the sensory organs, the hippocampus and the cortex and is

in charge of assigning emotional values to distinct stimuli (Sah et al. 2003). This will be the nuclei

group this dissertation focuses on.

1.7.2 Function

The amygdala is a part of the limbic system and is known to play a crucial role in processing

emotions and initiating fear and anxiety reactions (Trepel 2008). Functional loss of the amygdala

results in a decrease of negative emotions such as anxiety and rage (Weiskrantz 1956, Zola-

Introduction Hippocampus

11

Morgan et al. 1991). Concordantly, stimulation of the amygdala provokes anxiety and fear (Kaada

1951, Chapman et al. 1954). Furthermore, direct infusion of allopregnanolone into the amygdala

resulted in a decrease of anxiety-patterned behavior (Akwa et al. 1999).

1.8 Hippocampus

The hippocampus is an evolutionary old and well-conserved brain structure. It too is located in the

medial temporal lobe and belongs to the limbic system. It is a part of the hippocampal formation, a

group of functionally and neuronally connected cerebric areas, which in addition to the

hippocampus also includes the entorhinal cortex, the subiculum and the dentate gyrus. The actual

hippocampus equates to the Cornu Ammonis and can be functionally and morphologically

subdivided into 3 domains, the Cornu Ammonis 1 to 3 (CA1-CA3).

1.8.1 Neurophysiology

The hippocampus obtains information from several sensory systems. The entorhinal cortex, as the

main input source of the hippocampus, hereby channels said information (Lomo 1971). The

incoming data is then processed and forwarded back to the cortex. Within the neuronal network of

the hippocampal formation, there are three major successive excitatory projections, which

combined resemble the trisynaptic circuit (Doller et al. 1982, Yeckel et al. 1990). The starting point

is formed by the perforant path, which is made up of fibers emerging from the entorhinal cortex

leading to the dentate gyrus. From here the so-called mossy fibers innervate the CA3 region, which

in turn connects via the Schaffer collaterals to the CA1 region (Amaral 1993). Axons from the CA1

region then project to the subiculum and back to the entorhinal cortex, thus completing the

trisynaptic circuit (see Fig. 4). Within each region there are inhibitory GABAergic interneurons

processing the data (Jones 1993, Pettit et al. 2000). These GABAergic fibers are the working point

of GABAA receptor modulating drugs such as benzodiazepines.

Fig. 4: Schematic display of hippocampal

formation and the trisynaptic circuit

Modified according to (Amaral 1993). The hippocampal formation is made up out of the entorhinal cortex (EC), the dentate gyrus (DG), the hippocampus and the subiculum (SUB). The hippocampus can be subdivided into regions CA1-CA3. The trisynaptic circuit is a successive pathway that is made up of three major excitatory projections: the perforant path (pp) emerging from the EC to innervate the DG, the mossy fibers (mf) that connect the DG with the CA3 region of the hippocampus and the schaffer collaterals (sc) that excite the CA1 region

Introduction Thalamocortical Connections

12

1.8.2 Function

A core function of the hippocampus is the consolidation of the declarative memory. Scoville and

his team were the first to discover, that upon bilateral removal of the hippocampus, patients suffered

from anterograde amnesia (Scoville et al. 1957). Afterwards it was repeatedly confirmed that the

hippocampus is indeed crucial for forming new memories (Moss et al. 1981, Eichenbaum et al.

1992, Squire 1992). Close evaluation showed that hereby only the declarative memory was

compromised, whilst the procedural memory was not impaired (Eichenbaum et al. 1992). Since the

hippocampus plays such a leading role in memory formation it can be supposed, that drugs with

amnestic side effects interfere within some point of hippocampal data processing. For instance,

benzodiazepines might act at hippocampal GABAA receptors, enhancing inhibitory interneurons

that subsequently reduce data transfer and therefore prevent memory consolidation.

1.9 Thalamocortical Connections

The thalamus is densely connected to the cortex and together with the latter shapes up to form the

thalamocortical system (Guillery et al. 2002).

1.9.1 Anatomy and Neurophysiology

The thalamus is located in the diencephalon, can be anatomically split into a ventral and dorsal

thalamus and consists of numerous nuclei. The nuclei can be divided according to their

connections. While some nuclei (so called specific nuclei) send their efferent nerve fibers quite

distinct to each respective corresponding area of the cerebral cortex and gather information from

sensory and sensitive organs for the most part, others have rather fewer direct efferent nerve fibers

to the cortex but connect mainly to the first mentioned sort of thalamic nuclei (Trepel 2008). The

second kind of nuclei, also called unspecific nuclei, receive their afferent input from the cerebellum,

the basal ganglia, the cortex and above all from the reticular formation. Their efferent fibers to

specific nuclei serve to modulate the activity and therefore output of the specific nuclei (Trepel

2008).

A certain nucleus of the ventral thalamus needs to be emphasized: the thalamic reticular nucleus

(TRN) that wraps around the remaining thalamus (Fig. 5A). As indicated by the name, it mostly

consists of reticular cell that converse with diverse specific nuclei in an inhibitory manner using

GABAergic connections (Lubke 1993). It is reached by collaterals of thalamocortical and

corticothalamic fibers and regulates activity in the dorsal thalamus accordingly (Fig. 5B) (Jones

1975, Shosaku et al. 1989). The TRN is regulated itself by the reticular formation (located in the

brainstem) via inhibitory afferent fibers, which enables activity of thalamocortical fibers by

disinhibition, thus allowing efferent nerve fibers of specific thalamic nuclei to excite the cerebral

Introduction Thalamocortical Connections

13

cortex (Fig. 5B) (McCormick et al. 1997, Trepel 2008). While the excitatory cortex afferent fibers

quite distinguished and selective determine which specific nuclei will be increasingly inhibited by

the TRN, the inhibitory reticular formation afferents inhibit the TRN in an undirected manner (Trepel

2008).

The cerebral cortex plays a vital role in perception, awareness, attention, thought, intended actions

and language (Trepel 2008). The most part of the cortex is amounted by neocortex, the evolutionary

youngest developed component of the brain (Trepel 2008). Due to distribution of different cellular

types, it can be sectioned into six layers, also called laminas, which each play their role in neuronal

processing (Mountcastle 1997). Layer 4 is known to be the main entrance of external information

to the cerebral cortex, basically where specific thalamic nuclei efferent nerve fibers end (Trepel

2008). Neurons of the 5th layer project to diverse subcortical destinations except the thalamus

(Molnár et al. 2006). Lamina 6 is made up out of many different cells, whereupon the pyramidal

cells of this layer sent their axons to the specific nuclei of the thalamus (Trepel 2008). Layer 1 to 3

serve to modulate lower layers and to interconnect cortex areal units (Trepel 2008).

Fig. 5: Schematic sketch of the thalamocortical connections

A: Sketch of a coronary brain slice depicting the location of the thalamus and the cortex. Changed according to (Wahl-Schott et al. 2009). B: Simplified display of the thalamocortical connections. Hereby the + indicates glutamatergic fibers while the – represents GABAergic contacts. Ascending sensory information activates neurons of the specific thalamic nuclei, which then in turn excite the cortex. Thalamoreticular neurons modulate relay neuron function via GABAergic synapses. When the reticular formation as a part of the reticular activation system (RAS) is active, it inhibits inhibitory thalamoreticular neurons, disinhibiting negative influence of thalamoreticular neurons on thalamocortical (TC) neurons. Changed according to (Wahl-Schott et al. 2009, Mattusch 2012)

TC neuron

Thalamoreticular

neuron

Pyramidal

cell of cortex

RAS Sensory input

Cortex

Ncl. reticularis thalami

Thalamus

B

Introduction Aim of this dissertation

14

1.9.2 Function

In means of perception of one’s environment the thalamus plays the role of an operator. All sensory

information gathered, apart from olfaction, passes through the diverse thalamic nuclei, where it is

sorted and selected before being rendered to the respective cortex areas (McCormick et al. 1997,

Huguenard et al. 2007). This switchover serves as a filter, deciding which part of the incoming

information flow is relevant for the individual at the moment and therefore passed on to the cortex,

i.e. to consciousness, and vice versa. Thus did the thalamus acquire its reputation as the gate to

consciousness. In addition to its gate-like function, the thalamus also portrays a regulatory function

in alternating states of consciousness, such as sleeping and being awake (McCormick et al. 1997,

Steriade 2005). Therefore benzodiazepine effect on the thalamocortical connections may be

responsible for side effects such as drowsiness and sedation.

1.10 Aim of this dissertation

Benzodiazepines such as diazepam and the TSPO ligand XBD elicit similar anxiolytic properties

but feature diverging side effect profiles. While diazepam exposes amnestic and sedative adverse

effects as well as tolerance development and risk of addiction, XBD lacks any of those, as far as

experiments on humans and rodents have surveyed (Kita et al. 2009, Rupprecht et al. 2009, Rote-

Liste 2013, 2015). XBD enhances the synthesis of neurosteroids such as allopregnanolone, which

are presumably responsible for XBD effects (Rupprecht et al. 2009). Both, allopregnanolone and

diazepam are positive allosteric modulators (PAMs) at the GABAA receptor, though they occupy

different binding sides (Hosie et al. 2006, Möhler 2006). Due to diverse GABAA receptor subunit

expression, GABAA receptor binding abilities vary throughout different brain areas, probably

accounting for the discrepancy of diazepam and XBD side effect profile (see Fig. 6) (Pirker et al.

2000).

The intention of this study is to investigate the diverging side effect profile of diazepam and XBD

on a neurophysiological level using the imaging technique voltage sensitive dye imaging (VSDI).

Therefore, a series of experiments was designed and conducted to determine neuronal activity

propagation under influence of diazepam, as a representative of benzodiazepines, and XBD.

According to the different side effect profile of these drugs, VSDI experiments in areas of the brain

crucial for neuronal processing related to anxiety, memory and sedation, namely the amygdala,

hippocampus and thalamocortical region respectively were performed. To verify diazepam and

XBD effects, measurements with their antagonists (flumazenil and finasteride) were performed as

well.

Introduction Aim of this dissertation

15

Additionally, experiments with allopregnanolone, a neurosteroid acting as a PAM at GABAA

receptors and producing anxiolysis, were performed (Bitran et al. 1995). It has been shown that

allopregnanolone synthesis increases upon XBD-induced TSPO activation (Rupprecht et al. 2009).

Fig. 6: Scheme of brain regions associated with benzodiazepine effects and adverse effects

According to (Nothdurfter et al. 2012b). Depicted are the brain areas relevant for the adverse effect profile of benzodiazepines and neurosteroids based on the specific GABAA receptor selectivity (red: receptive for benzodiazepines, blue: receptive for neurosteroids) and GABAA receptor subunit distribution in the brain. The color coded chart indicates in which brain region the side effects origin:

Anxiety: cerebral cortex, amygdala (AM), bed nucleus of stria terminalis (BNST), hippocampus (HIP) (Charney et al. 1996, Brandao et al. 2003) Sedation: AM, formatio reticularis (FR) (Bonin et al. 2008) Hypnosis: ventrobasal nucleus (VB), reticular nucleus (RTN), locus coeruleus (LC), Raphe nuclei (RN), substantia nigra (SN), ventrolateral preoptic nucleus (VLPO), tuberomamillary nucleus (TMN) (Bonin et al. 2008) Abuse: N. accumbens (NA), ventral tegmental area (VTA) (Di Chiara et al. 1993) Physical dependence: NA, VTA (Lingford-Hughes et al. 2003, Wafford 2005) Tolerance: HIP, NA, AM (Wafford 2005) Withdrawal: HIP, NA, AM (Wafford 2005) Amnesia: cerebral cortex, HIP (Savic et al. 2005, Bonin et al. 2008)

Abbreviations: extra - extrasynaptical receptor, syn - synaptical receptor, IN – interneuron, PN - pyramidal neuron

The diagram is not suitable to compare the absolute levels of various subunits in either brain region. The distribution of GABA receptor mRNA is not indicated. (Nothdurfter et al. 2012b)

Material and Methods Preparation of brain slices

16

2 Material and Methods

To observe the effects of XBD, diazepam and allopregnanolone in the desired brain regions, brain

slices of mice were prepared. The respective slices were subsequently stained with a voltage

sensitive fluorescent dye, that allowed monitoring changes in signal transduction of neuronal

networks due to substance application (enhancement of GABAergic inhibition) using VSDI.

A detailed description of all applied solutions is listed in the appendix.

2.1 Preparation of brain slices

All experiments were performed on 3 to 8 week old male C57Bl6 mice. The rodents were

decapitated under sedation with the inhalation anesthetic Isofluran. All steps of the preparation

henceforth were realized in ice-cold sucrose-based-ACSF (artificial cerebrospinal fluid) saturated

with carbogen (95% oxygen / 5% carbon dioxide). Access to the brain was achieved after stripping

the skull by two transversal cuts from caudal and a shallow guided cut along the crown’s midline.

The divided cranium halves were bent upwards with forceps, the Nervi optici torn and the brain

carefully removed from the cranial cavity using a rounded spatula. Depending on the desired slice

the brain was prepared further differently. The obtained plain surface was then glued to a metal

block with a cyanoacrylate-glue (Histoacryl, B. Braun, Melsungen, Germany).

Coronary brain slices

In order to get a preparation of the amygdala, the cerebellum was cut off with a razor blade in a

coronary manner.

Sagittal brain slices

For preparation of sagittal brain slices to reach the hippocampus the brain was divided into its

hemispheres via a sagittal cut after removing the cerebellum.

Thalamocortical brain slices

The thalamocortical connections were obtained using a method developed by Agmon and Connors

(Agmon et al. 1991). This preparation allows preservation of the ventrobasal nucleus of the

thalamus, the barrel cortex (somatosensory cortex of rodents) and the fiber tract connecting them.

To achieve intact connections the forebrain needs to be cut off at specific angles (55° sagittal, 10°

horizontal).

Material and Methods Voltage sensitive dye imaging

17

Subsequently, regardless of the desired preparation, the metal block with the glued on brain was

integrated into a vibratome (HM 650 V, Microm International, Walldorf, Germany). The brain has

been cut into slices of 350 µm (amygdala, hippocampus) and of 550 µm thickness (thalamocortical

slices; Fig. 9, p. 21). With the wide end of a Pasteur pipette the slices were transferred into a

transportable chamber filled with general ACSF continuously saturated with carbogen. For the

following 30 minutes they were incubated in a 34°C hot water bath, before being dyed, to allow the

slices to rest and recover from the stress of slice preparation.

2.2 Voltage sensitive dye imaging

VSDI is an imaging technique that allows monitoring of neuronal electric networks in high temporal

and spatial resolution imaging down to 20-50 µm and a millisecond (Chemla et al. 2010). Offering

the great advantage of visualizing the processing of neuronal activity in real time, VSDI provides a

new perspective in the field of neuroscience. Key element to this technique is the voltage sensitive

dye (VSD), which infiltrates organic tissue and binds to the external surface of cell membranes

without interrupting their usual function (Loew 2015). In addition to fluorescent properties, voltage-

sensitive-dyes feature the ability to change its emission spectrum according to the surrounding

charge. Therefore changes in membrane voltage are displayed as changes in fluorescence. This

feature allows the fast monitoring of neuronal signal transduction. Validation and accuracy of VSDI

has been confirmed by close correlation of the optical signal of VSDI recordings and changes in

membrane potential measured via conventional electrophysiological means (Davila et al. 1973,

Salzberg et al. 1973, Morad et al. 1979, Windisch et al. 1985, Chien et al. 1991). Since VSDI was

first introduced in 1968 (Tasaki et al. 1968) it has further improved thanks to the development of

new and superior dyes and progress in technology.

2.2.1 Staining of brain slices with Di-4-ANEPPS

In this dissertation brain slices were stained with Di-4-ANEPPS ((4-(2-(6-(Dibutylamino)-2-

naphthalenyl)ethenyl)-1-(3-sulfopropyl)pyridinium hydroxide inner salt; Sigma-Aldrich Chemie

GmbH; Schnelldorf; Germany), a well-established and commonly used fast acting voltage sensitive

dye (Fluhler et al. 1985, Thiele 2008, Stepan et al. 2012), which allows the monitoring of voltage

changes within a millisecond range. When excited with light of wavelengths between 350 to 550

nm it emits light with longer wavelengths (about 500 to 800 nm) (stokes shift). If the surrounding

tissue is electrically excited the fluorescence shifts to lower wavelength due to a shift of the

absorption, excitation and emission spectrum caused by the dyes electrochromism characteristic

(Loew et al. 1992). Therefore differences of tissue charge can be measured by assessing the

intensity of emission equal and higher than a certain wavelength as demonstrated in the figure

below (Fig. 7). In addition due to the change of excitation spectrum when the surrounding tissue is

Material and Methods Voltage sensitive dye imaging

18

depolarized, the dye will be less excited (presupposed the same spectrum of light enlightens the

dye) and consequently a further reduction of emission of fluorescence occurs (see Fig. 7).

Fig. 7: Schematic sketch of the principle of voltage sensitive shift of emission

Modified according to (Loew et al. 1992). Depending on the voltage of the surrounding tissue the emission spectrum varies due to the electrochromism characteristic of voltage sensitive dyes. As depolarization advances the excitation and emission spectrum of the dye changes (depicted by the arrows in the middle diagram). In addition due to the change of excitation spectrum when the surrounding tissue is depolarized, the dye will be less excited (presupposed the same spectrum of light enlightens the dye) and consequently a further reduction of emission of fluorescence occurs (depicted by the arrow in the last diagram). Therefore changes in membrane voltage of the surrounding tissue can be calculated by considering the intensity of the fluorescence greater or equal of a certain wavelength (represented as the green area).

Depending on the investigated region of the brain and therefore variable necessity of tissue

magnification, the setup was build up differently, with or without a Barlow lens. Since the Barlow

lens also affected exposure to light, which in turn affected excitation and emission of the dye, two

different dyeing methods were applied.

Staining procedure for slices of the amygdala and thalamocortical connections

Di-4-ANEPPS, aliquoted in DMSO (10 mg/ml) was stored at -20°C. Before staining the dye was

incubated at 50° for 5 minutes and thoroughly vortexed. 7.5 µl were properly mixed in with 10 ml

-90 mV

0 mV

Material and Methods Voltage sensitive dye imaging

19

ACSF (final concentration of about 0.0075 mg/ml) and filled into a petri dish continually aerated

with carbogen. Here the slices were stained for 15 minutes in a shaded room.

Staining procedure for hippocampal slices

Initially 5 mg of Di-4-ANEPPS were aliquoted in 1 ml DMSO and 0.5 ml 10-%-El-Cremophor-

solution and kept at -20°C. For staining 40 µl of the aliquot were blended in with 0.5 ml ACSF and

0.5 ml fetal bovine serum. The slices were dyed in the resulting staining solution under continuous

aeration with carbogen for about 20 minutes in a darkened room.

After either staining procedure excessive dye was rinsed off with fresh ACSF. Following the slices

were transferred to a shaded, ACSF-filled, continuously carbogen-aerated holding chamber where

they were allowed to rest for at least 30 minutes before starting measurements. From this point on

and forward Bicuculline (GABAA-R-antagonist) was always added to the ACSF to reach a

concentration of 0.6 µMolar. This step was necessary to assure an adequate signal for two reasons

(Stepan et al. 2012). First, Di-4-ANEPPS potentiates GABA function at the GABAA-receptor

(Mennerick et al. 2010) and second due to their in comparison longer range, glutamatergic axons

are cut to a greater extend than the usually relatively shorter ranged axons of GABAergic

interneurons during brain slice preparation (Stepan et al. 2012).

2.2.2 VSDI Setup

The VSDI-Setup including recording chamber, microscope and electrodes was safely positioned

on a vibration-cushioned table (via pressurized air). To avoid electric noise it was encased by a

Faraday cage and earthed. The employed epifluorescence microscope (BX51 WI, Olympus,

Hamburg, Germany) was equipped with a MiCAM02 HR-CCD camera and processor (BrainVision

Inc., Tokyo, Japan) and an XLFluor4X/340 objective lens (NA 0.28; Olympus, Hamburg, Germany).

A motorized micromanipulator (Luigs and Neumann, Ratingen, Germany) was enabled to manage

movement of the microscope, making it possible to arrange it in all three dimensions without

affecting the recording chamber.

Source of light was a halogen bulb (MHAB-150 W; Moritex corp., China). A software-triggered

shutter controlled light exposure. A band-pass excitation filter ensured that only light of 480-550

nm wavelengths illuminated the slice. A dichroitic mirror (570 nm) reflected the light, projecting it

onto the slice. The fluorescence however, being of longer wavelength, passes through the dichroitic

mirror and through a following long-pass emission filter (590 nm). Thus ensuring that only the dye-

emitted fluorescence is detected by the CCD sensor chip and that the resulting signal is unaltered

by excitation radiation and potential other fluorescence (Fig. 8A). The recorded signal is then

processed as normalized differences of fluorescence intensity [∆F/F] in each pixel over time and

presented as a color-coded sequence-film on the monitor (Fig. 8B).

Material and Methods Voltage sensitive dye imaging

20

A B

Fig. 8: Schematic sketch of the VSDI-setup and presentation of the processed signal

A: schematic sketch of the VSDI-setup, slightly modified according to (Mühlpfordt et al. 2008). Bulb light was filtered by a band-pass excitation filter and projected onto the slice by a dichroitic mirror. Fluorescence emerging from the stained slice passed through the dichroitic mirror and through a following long pass emission filter to the CCD-sensor (detector). B: Color-coded presentation in BrainVision of the processed CCD-camera-recorded signal projected onto a live-picture of the employed brain slice.

2.2.3 Recordings

The slices were carefully positioned in the recording chamber and fixed by a grid (thin nylon threads

tensely attached to a platinum frame). Continuously carbonated ACSF perfused the recording

chamber throughout the entire experiment at a flow rate of about 5-8 ml/min. Experiments were

conducted at room temperature to increase tolerance of hypoxia. To monitor changes in signal

transduction of neuronal networks by GABAergic inhibition under substance influence, the slices

were artificially electrically excited. For excitation of the to examine brain regions a bipolar tungsten

electrode was carefully placed visually. For analysis only the desired regions were considered.

Therefore a ROI (region of interest) was placed to include the area under investigation.

Basolateral amygdala:

The electrode was cautiously positioned inside the amygdala right next to the branching point of

the external capsule that encloses the amygdala (Fig. 9A). The ROI was put to include the

basolateral amygdala.

CA1 region of the hippocampus:

In this preparation the Schaffer collateral was stimulated and the signal obtained from the CA1

region of the hippocampus innervated by it (Fig. 9B).

dF/Fmax (%)

Material and Methods Voltage sensitive dye imaging

21

Thalamocortical preparation:

In this slice preparation two electrodes were positioned (Fig. 9C), one in the ventrobasal thalamus

and another one where layer 4 of the cortex was suspected to be and a total of three areas were

investigated, including the thalamus, the thalamocortical connections and the cortex itself to not

only examine whether the substances have an effect but also to distinguish at which point the

thalamocortical connectivity is being manipulated. Therefore the ROI was positioned to include the

cortex in both stimulation techniques and the thalamus as a second area of interest when

stimulated inside the thalamus.

A B C

Fig. 9: Display of the slice preparations and electrode and ROI placement

White arrows indicate electrode placement. A: Amygdala preparation, the electrode was placed next to the branching point of the external capsule to stimulate the basolateral and lateral amygdala (yellow frame). B: Hippocampus preparation, the electrode was placed to excite the Schaffer collateral, which stimulate the CA1 region of the hippocampus (green). C: Thalamocortical preparation, two electrodes were placed, one in the thalamus to stimulate thalamocortical connections and one in the cortex. Region of interests were the cortex (blue) stimulated both ways and the thalamus (red) stimulated by the electrode placed in the thalamus.

The intensity and duration of pulse stimulation was adjusted via isolated stimulators (type 2533,

Devices Instruments, Welwyn Garden City, England). Single square pulses were applied to

determine the necessary stimulus intensity (0.1-100 V) and length (0.05-0.2 ms). They were set for

each experiment individually in order to generate a similar intense signal throughout all

experiments.

Recording settings were adjusted by BrainVision software (BrainVision 13.04.20, BrainVision Inc.,

Tokyo, Japan). 512 frames were recorded with a sample rate of 2.2 ms per frame, allowing a spatial

resolution of 88*60 pixel with a pixel size of 8.4*9.8 µm in the hippocampus preparations and

36.4*40.0 µm in the amygdala and thalamocortical slices. The resulting 512 images were put

together as a sequence to a film of 1.126 seconds. All electric stimuli were applied in the 103rd

Frame, except for the pulses set in the thalamus, which were released in the 48th Frame.

Recordings were set to record 16 sequences of 512 frames every 6.5 minutes. To improve signal

to noise ratio 16 recorded sequences were averaged, resulting in one averaged sequence every

Material and Methods Data analysis

22

6.5 minutes. This recording protocol was altered for thalamocortical slice measurements, since

there were two electrodes placed in this preparation. Alternating they stimulated the slice,

producing an averaged sequence made of 16 recordings each in a time frame of 11.5 minutes. The

protocol was controlled by WinLTP 1.11b software (Anderson and Collingridge, University Bristol,

UK), which triggered the stimulators and the shutter (controlling light exposure) according to the

set protocol. To spatially and temporally smooth ∆F/F values a 3x3x3 average filter was applied for

all quantifications in addition to an interpolation function to reduce pixilation of images.

As soon as all settings were adjusted measurements started. Once there has been a steady signal

in the desired region for at least 19 minutes (3 control movies), the investigated substance was

applied (XBD: 3 µM, diazepam: 1 µM, allopregnanolone: 1 µM) and recordings continued for about

an hour. Therefore, in addition to the 3 control recordings, 8 more recordings of substance influence

were achieved in amygdala and hippocampal slices or in case of thalamocortical preparation 5

recordings of substance influence. In experiments with diazepam flumazenil (10 µM) was added

afterwards to antagonize diazepam. In addition experiments with finasteride (10 µM) and XBD were

performed, to inhibit XBD effects. Different from flumazenil, finasteride needed to be applied at the

same time as XBD. XBD’s mode of action is via enhancement of neurosteroid genesis, which then

in turn carry out the actual impact. As a 5α-reductase-inhibitor, finasteride prevents neurosteroids

from being synthesized, but cannot antagonize effects of already synthesized neurosteroids. To

avoid data interference by possible effects of finasteride itself it was already added to the ACSF

during control recordings. Finally, to avoid interference by potential (though unlikely, considering

the small amounts) cell damage caused by the Alcohol (70%) used for XBD aliquotation, the same

amount of Alcohol was also added to the ACSF during control recordings in all experiments

containing XBD medication.

2.3 Data analysis

The measured data was presented in BrainVision as a color-coded signal in a sequence-film of

1.126 seconds. This signal can be broken down into three main components: Intensity, spread and

duration of the excitation. Therefore BrainVision-data was processed with a custom programmed

macro in Matlab (Matlab R2008b, The MathWorks, Natick, USA). Hereby the BrainVision acquired

fluorescence inside the ROI representing the fast depolarization signal (FDS) was broken down

into descriptive and comparable numerical values as described below:

The ‘area’ is a numeric value counting all active pixel. An active pixel is defined as a pixel inside

the ROI where the change of fluorescence (∆F/F) was at least three times higher than the standard

deviation of random background noise at anytime within the 512 frames. It is a parameter to

determine the spread of the excitation upon stimulation.

Material and Methods Data analysis

23

The ‘FDSmaxInt’ [∆F/F] is a value describing the mean of the maximal change of fluorescence

throughout all frames of every active pixel within a ROI.

The value ‘FDSareaInt’ [∆F/F] represents the sum of the intensities of every active pixel for each of

the 512 frames within a defined ROI. It will be displayed as a graph of FDSareaInt values over time.

Since neuronal activity propagation upon stimulus only lasts for about 50-300 ms, depending on

the brain area, and since the dye bleaches over time, only the first 228 frames (500 ms) will be

presented in this dissertation.

The ‘amplitude of FDSareaInt’ is a parameter that amounts to the maximal FDSareaInt value under

substance influence normalized to the maximal FDSareaInt value of the respective control recording,

basically representing the maximal amplitude of the FDSareaInt graph (peak of neuronal activity)

over the 550 frames. This parameter depicts the effect the substances have on propagation and

intensity of neuronal activity upon stimulation.

The product ‘FDSAUC’ [∆F/F] is a value designed to give an impression of a summarized overall

effect. It includes the compounds propagation (area), Intensity (Int) and duration of excitation. It is

calculated by adding the values of ‘FDSareaInt’ [∆F/F] of all frames from pulse release till excitation

fade-out, basically resembling the area under the curve (AUC) of the graph depicting the FDSareaInt.

To allow comparability the data was normalized to its respective control (all values were divided by

the mean value of the last 19.8 ms before electric stimulation of the respective three controls). The

results are present by comparing the last recording of the substance to the last control recording.

They will be featured as mean ± SEM (standard error of the mean). Diagrams were created in Excel

(Excel 2003, Microsoft Corporation, Redmond, USA).

Statistic significance was investigated with either the Wilcoxon signed rank test or the Man-

Whitney-U test depending on pairing. Level of significance was defined as α = 0.05 and is indicated

by asterisks as followed: * for p < 0.05, ** for p < 0.02 and *** for p < 0.01.

Results Alteration of neuronal activity upon stimulus in the Amygdala by diazepam, XBD and

allopregnanolone

24

3 Results

Several rows of experiments in the amygdala, the hippocampus and the thalamocortical

connections were performed as described above. In the figure below an exemplary excitation

transmission of neuronal activity in the cortex upon thalamic stimulation is depicted.

Fig. 10 Excitation transmission in the thalamocortical circuit

Depicted is the thalamocortical cut, the first picture is before stimulation (A), then stimulus is released by the electrode in the thalamus (B). The stimulus spreads throughout the thalamus (C) before exciting the thalamocortical connections and hitting the cortex (D) where it spreads (E-F) before fading away again (G-H).

3.1 Alteration of neuronal activity upon stimulus in the Amygdala by diazepam,

XBD and allopregnanolone

Since the amygdala is the crucial area for those information processing related to fear and anxiety

and hence, initiates referring actions (see ch. 1.7) a set of experiments was performed in this brain

region.

3.1.1 Effect of diazepam on neuronal activity upon stimulus in the amygdala

Diazepam application to amygdala brain slices resulted in a decrease of the stimulated excitation

transmission in the basolateral amygdala. As depicted in the graph below (Fig. 11A), FDSareaInt

showed a decrease throughout stimulated excitation transmission by diazepam (amplitude of

FDSareaInt 81.0 ± 8.2 %, n=6). FDSAUC showed a distinct though not significant decrease to 85.0 ±

12.8 % (n=6) in average. Hereby especially the FDSmaxInt of neuronal activity upon stimulus was

reduced (82.2 ± 0.05 %, n=6, p<0.05) (Fig. 11B), while the area remained quite stable (98.2 ± 6.6

%, n=6) (Fig. 11C). The subsequent application of the benzodiazepine antagonist flumazenil

A B C D

E F G H

dF/Fmax

(%)

Results Alteration of neuronal activity upon stimulus in the Amygdala by diazepam, XBD and

allopregnanolone

25

resulted in an increase of the diminished neuronal activity (FDSAUC 100.6 ± 12.8 %, n=6),

successfully though not significantly antagonizing diazepam effect.

A

B C

Fig. 11: Diazepam impact on neuronal activity in the amygdala

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and diazepam (red) recordings. On the right, the maximal normalized FDSareaInt values of control and diazepam (diaz, 0.81 ± 0.08, n=6) and 10 µg flumazenil (fluma, 0.92 ± 0.08, n=6) recordings in the amygdala are depicted, representing the normalized amplitude of their peak values. B: Effect of 1 µM diazepam (red) on FDSmaxInt (∆F/F) of the VSDI-signal in the amygdala. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. Distinct and significant decrease of FDSmaxInt by diazepam 0.82 ± 0.05 (n=6, p<0.5). C: No significant alteration of area in the amygdala under 1 µM diazepam 0.98 ± 0.07 (n=6).

3.1.2 Effect of XBD on neuronal activity upon stimulus in the amygdala

XBD application to amygdala brain slices resulted in a decrease of the stimulated excitation

transmission in the basolateral amygdala. As depicted in the graph below (Fig. 12A), FDSareaInt

showed a decrease throughout stimulated excitation transmission by XBD (amplitude of FDSareaInt

0,4

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a

Results Alteration of neuronal activity upon stimulus in the Amygdala by diazepam, XBD and

allopregnanolone

26

83.3 ± 4.8 %, n=6, p<0.05). FDSAUC showed a distinct though not significant decrease to 86.3 ± 8.5

% (n=6) in average. Hereby especially the FDSmaxInt of neuronal activity upon stimulation was

reduced (87.4 ± 2.3 %, n=6, p<0.05) (Fig. 12B), while the area remained quite stable (95.6 ± 2.9%,

n=6) (Fig. 12C). XBD effect could not be significantly antagonized by joint application with

finasteride (amplitude of FDSareaInt 87.9 ± 7.0, n=6).

A

B C

Fig. 12: XBD impact on neuronal activity in the amygdala

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and XBD (green) recordings. On the right, the maximal normalized FDSareaInt values of, XBD (0.83 ± 0.05, n=6, p<0.05) and XBD plus finasteride (x/fina, 0.88 ± 0.07, n=6) recordings in the amygdala are depicted, representing the normalized amplitude of peak values. The black bar represents both control recordings. B: Effect of 3 µM XBD on FDSmaxInt (∆F/F) of the VSDI-signal in the amygdala. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. Distinct and significant decrease of FDSmaxInt by XBD 0.87 ± 0.03 (n=6, p<0.05). C: No significant alteration of area in the amygdala under 3 µM XBD influence 0.96 ± 0.03 (n=6).

0,4

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orm

. am

plit

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e o

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Results Alteration of neuronal activity upon stimulus in the Amygdala by diazepam, XBD and

allopregnanolone

27

3.1.3 Effect of allopregnanolone on neuronal activity upon stimulus in the amygdala

Allopregnanolone application to amygdala brain slices resulted in a decrease of the stimulated

excitation transmission in the basolateral amygdala (Fig. 13A). As depicted in the graph below (Fig.

13A), FDSareaInt showed a decrease throughout stimulated excitation transmission by

allopregnanolone (amplitude of FDSareaInt 88.9 ± 3.8 %, n=6, p<0.05). FDSAUC showed a distinct

though not significant decrease to 80.5 ± 6.0 % (n=6) in average. Hereby especially the FDSmaxInt

of neuronal activity upon stimulus was reduced (87.6 ± 4.3 %, n=6, p<0.05) (Fig. 13B), while the

area remained quite stable (99.1 ± 5.1 %, n=6) (Fig. 13C).

A

B C

Fig. 13: allopregnanolone impact on neuronal activity in the amygdala

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and allopregnanolone (yellow) recordings. On the right, the maximal normalized FDSareaInt values of control and allopregnanolone (allo, 0.89 ± 0.04, n=6, p<0.05) recordings in the amygdala are depicted, representing the normalized amplitude of peak values. B: Effect of 1 µM allopregnanolone on FDSmaxInt (∆F/F) of the VSDI-signal in the amygdala. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. Distinct and significant decrease of FDSmaxInt by allopregnanolone 0.88 ± 0.04 (n=6, p<0.05). C: No significant alteration of area in the amygdala under 1 µM allopregnanolone 0.99 ± 0.05 (n=6).

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Results Alternation of neuronal activity upon stimulus in the hippocampus by diazepam, XBD and

allopregnanolone

28

3.1.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in

the amygdala

A

B

C

Fig. 14 Comparison of substance effects on FDSAUC, FDSmaxInt and area in the amygdala

Bar chart depicting diazepam (red), XBD (green) and allopregnanolone (yellow) effect on FDSmaxInt (A), area (B) and FDSAUC (C) in the amygdala. The rounded values are displayed on the bottom of the bar chart, since they are each normalized to their respective controls, the value of each control is set at 1.00 and only displayed once, representing all controls. Fault indicators demonstrate standard error of the mean, for exact values see chapter 3.1. Asterisks illustrate significant change between the substance and it’s respective control.

3.2 Alternation of neuronal activity upon stimulus in the hippocampus by

diazepam, XBD and allopregnanolone

As the brain area that is essentially involved in memory formation, the hippocampus portrays the

brain area the next set of experiments was realized in.

3.2.1 Effect of diazepam on neuronal activity upon stimulus in the hippocampus

Diazepam application to hippocampal brain slices resulted in a decrease of the stimulated exci-

tation transmission in the basolateral amygdala (FDSAUC 82.2 ± 7.90 %, n=7). As depicted in the

1,00 0,82 0,87 0,880

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1,00 0,98 0,96 0,990

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ized

FD

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C

Results Alternation of neuronal activity upon stimulus in the hippocampus by diazepam, XBD and

allopregnanolone

29

graph below (Fig. 15A), FDSareaInt showed a distinct decrease throughout stimulated excitation

transmission by diazepam (amplitude of FDSareaInt 84.4 ± 4.5 %, n=7, p<0.02). Hereby intensity of

neuronal activity upon stimulation was distinctly though not significantly reduced in average to 90.4

± 4.2 % (n=7) (Fig. 15B). The decrease in area was significant and averaged to 88.0 ± 4.1 % (n=7,

p<0.02) of initial stimulus propagation (Fig. 15C). Subsequent admission of benzodiazepine

antagonist flumazenil resulted in significant increase of the diminished excitation transmission (area

= 101.3 ± 2.7 %, n=7, p<0.05), however increase of iazepam-diminished FDSmaxInt (91.6 ± 6.4 %,

n=7) and FDSAUC (85.6 ± 8.0, n=7) by flumazenil was only slight and not significant.

A

B C

Fig. 15: diazepam impact on neuronal activity in the hippocampus

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and diazepam (red) recordings. On the right, the maximal normalized FDSareaInt values of control and diazepam (diaz, 0.84 ± 0.04, n=7, p<0.02) and 10 µg flumazenil (fluma, 0.91 ± 0.07, n=7) recordings in the hippocampus are depicted, representing the normalized amplitude of their peak values. B: Effect of 1 µM diazepam (red) on FDSmaxInt (∆F/F) of the VSDI-signal in the hippocampus. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. Distinct decrease of intensity by diazepam 0.90 ± 0.04 (n=7). C: Effect on the area in the hippocampus. Significant reduction of area under 1 µM diazepam 0.88 ± 0.04 (n=7, p<0.02).

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Results Alternation of neuronal activity upon stimulus in the hippocampus by diazepam, XBD and

allopregnanolone

30

3.2.2 Effect of XBD on neuronal activity upon stimulus in the hippocampus

XBD application to hippocampal brain slices did not result in a significant alteration of the stimulated

excitation transmission in the basolateral amygdala (FDSAUC = 96.1 ± 7.8 %, n=7) (Fig. 16A).

Neither amplitude of FDSareaInt (93.2 ± 4.2 %, n=7), FDSmaxInt (93.5 ± 4.8 %, n=7) nor the area

(103.5 ± 4.8 %, n=7) of neuronal activity upon stimulation showed significant alteration under XBD

influence (Fig. 16B,C).

A

B C

Fig. 16: XBD impact on neuronal activity in the hippocampus

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 frames of control (black) and XBD (green) recordings. On the right, the maximal normalized FDSareaInt values of control and XBD (0.93 ± 0.04, n=7) recordings in the hippocampus are depicted, representing the normalized amplitude of their peak values. B: Effect of 3 µM XBD (green) on FDSmaxInt (∆F/F) of the VSDI-signal in the hippocampus. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. No significant decrease of FDSmaxInt by XBD 0.94 ± 0.05 (n=7). C No significant alteration of area under 3 µM XBD influence in the hippocampus 1.04 ± 0.05 (n=7).

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Results Alternation of neuronal activity upon stimulus in the hippocampus by diazepam, XBD and

allopregnanolone

31

3.2.3 Effect of allopregnanolone on neuronal activity upon stimulus in the hippocampus

Allopregnanolone application to hippocampal brain slices resulted in a decrease of the stimulated

excitation transmission in the basolateral amygdala (Fig. 17A). As depicted in the graph below (Fig.

17A), FDSareaInt showed a distinct decrease throughout stimulated excitation transmission by

allopregnanolone (amplitude of FDSareaInt 79.5 ± 4.1 %, n=6, p<0.05). FDSAUC showed a distinct

and significant decrease to 77.2 ± 5.0 % (n=6, p<0.05) in average. Hereby only the intensity of

neuronal activity upon stimulation was reduced (82.0 ± 3.0 %, n=6, p<0.05) (Fig. 17B), while the

area of activity propagation remained quite stable (97.1 ± 2.2 %, n=6) (Fig. 17C).

A

B C

Fig. 17: allopregnanolone impact on neuronal activity in the hippocampus

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 frames of control (black) and allopregnanolone (yellow) recordings. On the right, the maximal normalized FDSareaInt values of control and allopregnanolone (allo, 0.80 ± 0.04, n=6, p<0.05) recordings in the hippocampus are depicted, representing the normalized amplitude of their peak values. B: Effect of 1 µM allopregnanolone (yellow) on FDSmaxInt (∆F/F) of the VSDI-signal in the hippocampus. Control recordings are illustrated as black squares. The grey arrow indicates the average effect. Distinct and significant decrease of FDSmaxInt by allopregnanolone 0.82 ± 0.03 (n=6, p<0.05). C: Effect of 1 µM allopregnanolone on area of stimulus propagation in the amygdala. No significant alteration of area under allopregnanolone 0.97 ± 0.02 (n=6).

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Results

32

3.2.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in

the hippocampus

A

B

C

Fig. 18 Comparison of substance effects on FDSAUC, FDSmaxInt and area in the hippocampus

Bar chart depicting diazepam (red), XBD (green) and allopregnanolone (yellow) effect on FDSmaxInt (A), area (B) and FDSAUC (C) in the hippocampus. The rounded values are displayed on the bottom of the bar chart, since they are each normalized to their respective controls, the value of each control is set at 1.00 and only displayed once, representing all controls. Fault indicators demonstrate standard error of the mean, for exact values see chapter 3.2. Asterisks illustrate significant change between the substance and it’s respective control (one asterisks indicates p<0.05, double asterisks indicate p<0.02).

1,00 0,90 0,94 0,820

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Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

33

3.3 Alternation on neuronal activity upon stimulus in the thalamocortical

connections by diazepam, XBD and allopregnanolone

Two questions were addressed with the measurement in the thalamocortical connections. First, is

there an effect of the corresponding substances and second, if so, at which point within the

thalamocortical system does the effect occur: right in the thalamus, the connecting TC neurons or

within the cortex? Therefore, measurements were designed to detected neuronal activity in the

thalamus (intrathalamic measurements) as well as in the cortex (thalamocortical measurements)

upon stimulation in the ventrobasal thalamus. In addition FDS propagation in the cortex was

measured upon intracortical stimulation (intracortical measurements). Therefore an electrode was

additionally placed in layer 4 of the cortex.

3.3.1 Effect of diazepam on neuronal activity upon stimulus in the thalamocortical

connections

Diazepam application to thalamocortical brain slices resulted in a decrease of the FDS propagation.

As depicted in the graph below (Fig. 19A), FDSareaInt showed a distinct decrease of neuronal activity

in the cortex upon thalamic stimulation (amplitude of FDSareaInt 79.5 ± 3.2 %, n=7, p<0.02; FDSAUC

= 76.3 ± 4.9, n=7, p<0.02). Hereby especially FDSmaxInt of neuronal activity upon stimulation was

significantly reduced in average to 84.4 ± 3.11 % (n=7, p<0.02) (Fig. 19B). There was no significant

alteration in area (94.6 ± 3.01 %, n=7) (Fig. 19C). Subsequent admission of benzodiazepine

antagonist flumazenil resulted in distinct increase of the reduced area (FDSmaxInt = 96.0 ± 6.3 %,

n=7; area= 98.9 ± 2.9 %, n=7) (Fig. 19A). Intrathalamic (amplitude of FDSareaInt 87.7 ± 2.4 %, n=7,

p<0.02; FDSAUC = 86.4 ± 4.0 %, n=7) as well as intracortical (amplitude of FDSareaInt 83.3 ± 3.6 %,

n=6, p<0.05; FDSAUC = 81.4 ± 4.6 %, n=6, p<0.05) experiments with diazepam showed similar

results (Fig. 19B,C). Hereby diazepam showed equal impact on thalamocortical and intracortical

excitation propagation, while diazepam effect on the intrathalamic connections was slighter in

comparison. Intrathalamic diazepam impact could not be antagonized by subsequent flumazenil

application, in contrast to thalamocortical and intracortical diazepam effect.

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

34

A

B

C

Fig. 19: diazepam impact on neuronal activity in the thalamocortical connections

A: On the left averaged normalized FDSareaInt values of the first 500 ms of control (black) and diaze-pam (red) recordings are depicted. On the right, the maximal normalized FDSareaInt values of control and diazepam (diaz, 0.80 ± 0.03, n=7, p<0.02) and 10 µg flumazenil (fluma, 0.93 ± 0.07, n=7) recordings in the thalamocortical connections are depicted, representing the normalized amplitude of their peak values. B: Effect of 1 µM diazepam (red) on FDSmaxInt (∆F/F) of the VSDI detected signal in the thalamus (0.90 ± 0.02, n=7, p<0.02), the thalamocortical connections (0.84 ± 0.03, n=7, p<0.02) and intracortical (0.86 ± 0.03, n=6, p<0.05). Control recordings are illustrated as black squares. The grey arrow indicates the average effect. C: Effect of 1 µM diazepam on the area of stimulus propagation in the thalamus (0.94 ± 0.02, n=7, p<0.05), the thalamocortical connections (0.95 ± 0.03, n=7) and intracortical (0.92 ± 0.05, n=6).

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Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

35

3.3.2 Effect of XBD on neuronal activity upon stimulus in the thalamocortical connections

XBD application to thalamocortical brain slices resulted in a decrease of the stimulated excitation

transmission. As depicted in the graph below (Fig. 20A), FDSareaInt showed a distinct decrease of

neuronal activity in the cortex upon thalamic stimulation (amplitude of FDSareaInt 83.5 ± 3.2 %, n=8,

p<0.01; FDSAUC 77.6 ± 8.1%, n=8, p<0.02). Hereby especially FDSmaxInt of neuronal activity upon

stimulation was significantly reduced in average to 86.1 ± 3.3 % (n=8, p<0.01) (Fig. 20B), while

there was no significant alteration in area (102.0 11.5 %, n=8) (Fig. 20C). In experiments with

simultaneous admission of XBD and finasteride, no alteration of excitation propagation was

detectable (FDSmaxInt 101.8 ± 5.3 %, n=7; area 98.9 ± 2.9 %, n=7). Intrathalamic (amplitude of

FDSareaInt 86.2 ± 3.0 %, n=8, p<0.01; FDSAUC 84.2 ± 7.0 %, n=8) as well as intracortical experiments

(amplitude of FDSareaInt 87.4 ± 2.3 %, n=8, p<0.01; FDSAUC 84.6 ± 3.3 %, n=8, p<0.01) with XBD

showed similar results (Fig. 20B,C). Hereby XBD showed equal impact on thalamocortical and

intrathalamic neuronal activity upon stimulation, while intracortical XBD effect was slighter in

comparison. Intrathalamic and intracortical XBD impact could not be antagonized by additional

finasteride application, in contrast to thalamocortical XBD effect.

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

36

A

B

C

Fig. 20: XBD impact on neuronal activity upon stimulus in the thalamocortical circuit

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and XBD (green) recordings. On the right, the maximal normalized FDSareaInt values of XBD (0.83 ± 0.03, n=8, p<0.01) and XBD plus finasteride (X/Fina, 1.00 ± 0.05, n=7) recordings in the thalamocortical connections are depicted, representing the normalized amplitude of their peak values. The black bar represents both their respective control recordings. Additional finasteride application significantly antagonized XBD effects (p<0.05). B: Effect of 3 µM XBD (green) on FDSmaxInt of the VSDI-signal in the thalamus (0.89 ± 0.03, n=8, p<0.02), the thalamocortical connections (0.86 ± 0.03, n=8, p<0.01) and intracortical (0.93 ± 0.03, n=8). Control recordings are illustrated as black squares. The grey arrow indicates the average effect. C: Effect of 3 µM XBD on the area in the thalamus (0.94 ± 0.05, n=8), the thalamocortical connections (1.02 ± 0.11, n=8) and intracortical (0.96 ± 0.04, n=8).

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

Control XBD

no

rmal

ized

are

a

Intrathalamic

Control XBD

Thalamocortical

Control XBD

Intracortical

0,4

0,6

0,8

1

1,2

1,4

Control XBD

no

rmal

ized

FD

S max

Int

Intrathalamic

Control XBD

Thalamocortical

Control XBD

Intracortical

0

1

2

3

4

5

6

7

2,2 222,2 442,2

no

rmal

ized

FD

S are

aIn

t

time [ms]

Thalamocortical

0,83 1,000

0,2

0,4

0,6

0,8

1

1,2

Control XBD X/Fina

no

rm. a

mp

litu

de

of

FDS a

reaI

nt

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

37

3.3.3 Effect of allopregnanolone on neuronal activity upon stimulus in the thalamocortical

connections

As Fig. 21A demonstrates, allopregnanolone affected the duration of neuronal activity in the cortex

upon thalamic stimulation, as quantified by the parameter FDSAUC = 87.5 ± 9.4 % (n=5) in

comparison to amplitude of FDSareaInt (1.01 ± 6.5 %, n=5). Allopregnanolone application did not

affect FDSmaxInt of intrathalamic (97.6 ± 4.7 %, n=4) and thalamocortical (97.5 ± 5.4 %, n=5)

excitation transmission (Fig. 21B). However, allopregnanolone exertion resulted in decrease of

FDSmaxInt of intracortical neuronal activity upon cortical stimulation to about 81.5 ± 6.7 % (n=5).

Intrathalamic no alteration of area could be detected under allopregnanolone influence (93.8 ± 5.4

%, n=4). Allopregnanolone showed a tendency to increased thalamocortical (113.1 ± 13.3 %, n=5)

and intracortical stimulus propagation (area = 118.6 ± 10.8 %, n=5) (Fig. 21C). None of the

allopregnanolone experiments performed on thalamocortical slices provided significant results.

A

B

0

2

4

6

8

10

12

14

2,2 222,2 442,2

no

rmal

ized

FD

S are

aIn

t

time [ms]

Thalamocortical

1,010

0,2

0,4

0,6

0,8

1

1,2

Control Allo

no

rm. a

mp

litu

de

of

FDS a

reaI

nt

0,4

0,6

0,8

1

1,2

1,4

Control Allo

no

rmal

ized

FD

S max

Int

Intrathalamic

Control Allo

Thalamocortical

Control Allo

Intracortical

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

38

C

Fig. 21: allopregnanolone impact on neuronal activity upon stimulus in the thalamocortical connections

A: The diagram on the left shows averaged normalized FDSareaInt values of the first 500 ms of control (black) and allopregnanolone (yellow) recordings. On the right, the maximal normalized FDSareaInt values of control and allopregnanolone (allo, 1.01 ± 0.07, n=5) recordings in the thalamocortical connections are depicted, representing the normalized amplitude of their peak values. B: Effect of 1 µM allopregnanolone (yellow) on FDSmaxInt (∆F/F) of the VSDI-signal in the intrathalamic (0.98 ± 0.05, n=4), the thalamocortical connections (0.97 ± 0.05, n=5) and intracortical (0.81 ± 0.07, n=5). Control recordings are illustrated as black squares. The grey arrow indicates the average effect C: Effect of 1 µM allopregnanolone on the area of the stimulus propagation in the thalamus (0.94 ± 0.05, n=4), the thalamocortical connections (1.13 ± 0.13, n=5) and intracortical (1.19 ± 0. 11, n=5).

3.3.4 Comparison of diazepam, XBD and allopregnanolone effect on neuronal activity in

the thalamocortical connections

A

Control Allo

Thalamocortical

0,4

0,6

0,8

1

1,2

1,4

1,6

Control Allo

no

rmal

ized

are

aIntrathalamic

Control Allo

Intracortical

1,00 0,90 0,89 0,980

0,2

0,4

0,6

0,8

1

1,2

Co Di XBD Al

no

rmal

ized

FD

S max

Int

Intrathalamic

1,00 0,84 0,86 0,97

Co Di XBD Al

Thalamocortical

1,00 0,86 0,93 0,81

Co Di XBD Al

Intracortical

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

39

B

C

Fig.

22 Comparison of substance effects on FDSAUC FDSmaxInt and area in the thalamocortical connections

The bar charts serve to ease comparison of average effect of diazepam (red), XBD (green) and allopregnanolone (yellow) on FDSmaxInt (A), area (B) and FDSAUC (C) in the thalamocortical connections. The rounded values are displayed on the bottom of the bar chart, since they are each normalized to their respective controls, the value of each control is set at 1.00 and only displayed once, representing all controls. Fault indicators demonstrate standard error of the mean, for exact values see chapter 3.3. Asterisks illustrate significant change between the substance and it’s respective control (one asterisks indicates p<0.05, double asterisks indicate p<0.02).

1,00 0,94 0,94 0,940

0,2

0,4

0,6

0,8

1

1,2

1,4

Co Di XBD Al

no

rmal

ized

are

a

Intrathalamic

1,00 0,95 1,02 1,13

Co Di XBD Al

Thalamocortical

1,00 0,92 0,96 1,19

Co Di XBD Al

Intracortical

1,00 0,76 0,78 0,870

0,2

0,4

0,6

0,8

1

1,2

Co Di XBD Al

Thalamocortical

1,00 0,86 0,84 0,980

0,2

0,4

0,6

0,8

1

1,2

Co Di XBD Al

no

rmal

ized

FD

S AU

C

Intrathalamic

1,00 0,81 0,85 0,81

Co Di XBD Al

Intracortical

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

40

3.3.5 Analyses of diazepam, XBD and allopregnanolone impact on the individual layers of

the cortex

To get more insight into the diazepam, XBD and allopregnanolone mediated impact on

thalamocortical excitation propagation upon stimulation, the change in neuronal activity of each

layer of the cortex inside the ROI upon thalamic stimulation was investigated. For this purpose the

following parameters were analyzed in each layer individually: the maximal change of intensity

(Intmax) and the time till Intmax (delay of Intmax).

The reduction of Intmax by diazepam, XBD and allopregnanolone was distinct throughout all three

substances, however different amongst them in regard of the extend with which each layer was

affected (see

Fig. 24). Diazepam elicited most effect in layer 1 to 3 and was successfully and partially significantly

antagonized by flumazenil. XBD affected layer 1,2,5 and 6 the most. Allopregnanolone seemed to

diminish the maximal intensity of thalamocortical stimulus propagation least of all three substances

and only showed consistent decrease in layer 3 and 4.

Diazepam significantly decreased the delay of Intmax in the 2nd layer (74.7 ± 7.2 %, n=7, p<0.05)

and 4th layer (86.6 ± 4.8 %, n=7, p<0.05) and showed a tendency to decrease delay of Intmax in the

1st layer (89.5 ±7.7 %, n=7) without significance. Flumazenil did not significantly antagonize

diazepam effect on the delay of Intmax. XBD significantly decreased the delay of Intmax in the 4th

layer of the cortex (to 73.4 ± 13.4%, n=8, p<0.05). Simultaneous application of XBD and finasteride

showed to the tendency to but did not significantly prevent XBD effect in the 4th layer of the cortex.

Allopregnanolone application showed the same tendency in the 4th layer without significance (76.8

± 27.9 %, n=5). Apart form the 4th layer and in the case of diazepam also in the 1st and 2nd layer of

the cortex, the evaluation of delay of Intmax did not offer valuable information due to discrepancies

in the results.

Fig. 23 Display of cortex division

for layer analysis

Depiction of the roi (yellow outlining) and the 6 areas (I-VI) presenting the 6 lawyers of the cortex.

Results Alternation on neuronal activity upon stimulus in the thalamocortical connections by diazepam,

XBD and allopregnanolone

41

Fig. 24 Layer analysis of diazepam and XBD impact on normalized maximal Intensity

Depicted is a bar chart of the effect of diazepam (red), XBD (green) and allopregnanolone (yellow) on maximal Intensity normalized to their respective control recordings (black) in each layer (L1-6) of the cortex upon thalamic stimulation. Diazepam and XBD effects are depicted with their respective antagonists. The values are depicted inside the bars. Fault indicators demonstrate standard error of the mean. Asterisks represent significant results (one asterisks: p<0.05, two asterisks: p<0.02).

0,931

0,938

0,848

0,910

0,981

0,984

0 0,2 0,4 0,6 0,8 1 1,2

L1

L2

L3

L4

L5

L6

Kontrolle 1 µM AllopregnanoloneControl

normalized maximal Intensity

0,696

0,795

0,831

0,924

0,890

0,852

0,953

0,957

0,962

1,039

0,902

0,881

0 0,5 1 1,5

L1

L2

L3

L4

L5

L6

Kontrolle 1 µM Diazepam 10 µM FlumazenilControl

0,794

0,803

0,960

0,934

0,859

0,822

0,835

0,905

0,905

1,026

0,953

1,012

0 0,5 1 1,5

L1

L2

L3

L4

L5

L6

Kontrolle 3 µM XBD 10 µM FinasterideControl

normalized maximal Intensity normalized maximal Intensity

Discussion Result interpretation

42

4 Discussion

4.1 Result interpretation

Benzodiazepines such as diazepam and XBD, a ligand of TSPO, elicit similar anxiolytic properties

but dispose of diverging side effect profiles. While diazepam exposes amnestic and sedative

adverse effects as well as tolerance development and risk of addiction, XBD lacks any of those, as

far as experiments on humans and rodents have surveyed (Kita et al. 2009, Rupprecht et al. 2009,

Rote-Liste 2013, 2015). XBD enhances neurosteroid genesis, e.g. allopregnanolone synthesis,

which is responsible for XBD effects (Rupprecht et al. 2009). Both, allopregnanolone and diazepam

are positive allosteric modulators at the GABAA receptor, though they occupy different binding sides

(Hosie et al. 2006, Möhler 2006). Due to diverse GABAA receptor subunit expression, GABAA

receptor binding abilities vary throughout different brain areas, probably accounting for the

discrepancy of diazepam and XBD side effect profile (Pirker et al. 2000). To investigate diazepam,

XBD and allopregnanolone impact on neuronal activity in brain areas relevant to their effects,

alteration of excitation propagation upon stimulation under their influence was detected using VSDI

in the course of this dissertation. Brain areas included in the experiments are the amygdala as the

anxiety-center of the brain (see ch. 1.7), the hippocampus as the brain area where memory

formation occurs (see ch. 1.8) and the thalamocortical system, indispensable for awareness (see

ch. 1.9). Corresponding mice brain slices were prepared for VSDI and signal propagation upon

stimulus under the influence of diazepam, allopregnanolone and XBD was determined.

4.1.1 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the amygdala

The amygdala has been found to be crucially involved in processing emotions, especially anxiety

(see chapter 1.7.2). Diazepam, XBD and allopregnanolone displayed significant negative impact

on neuronal excitability in the amygdala. Hereby all three substances demonstrated similar

inhibition of signal transduction in the amygdala, whereupon especially the Intensity (FDSmaxInt)

rather than the area of signal propagation was affected, indicating that the amount of neurons

activated upon stimulation remained the same but the level of their excitation was reduced by

diazepam, XBD and allopregnanolone.

These results are in line with clinical observations and animal models determining their anxiolytic

potential. Similar anxiolytic properties of XBD and benzodiazepines have been observed in rodents

as well as in humans (Kita et al. 2009, Rupprecht et al. 2009). Comparable anxiolytic properties of

allopregnanolone have been shown in rodents as well (Bitran et al. 1991, Picazo et al. 1995).

Taken together, the anxiolytic effect of all three substances probably origins in their diminishing

effect on neuronal activity in the amygdala. In terms of similarity of XBD and allopregnanolone

Discussion Result interpretation

43

results, it is likely that the impact of XBD on the amygdala is due to allopregnanolone formation.

These results therefore support the data of Rupprecht at al (Rupprecht et al. 2009), which proposes

that XBD effects are mediated by allopregnanolone.

4.1.2 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the

hippocampus

Diazepam displayed distinct impact on neuronal activity in the hippocampus, whereupon the area

of the stimulation was significantly reduced and the intensity distinctly diminished, indicating that

diazepam did not only affect the amount of neurons excited upon stimulation but also reduced the

level of excitation. This appears to explain diazepam’s known adverse effect on memory resulting

in anterograde amnesia (Rote-Liste 2013, 2015). XBD however, did not significantly disturb

neuronal activity upon stimulus in the hippocampus, indicating that XBD administration to subjects

would not result in affection of memory. Just as no affection in memory upon XBD application has

been observed in clinical studies so far (Rupprecht et al. 2009).

Interestingly, allopregnanolone had a significant negative effect on the intensity of signal

transduction in the hippocampus even though XBD showed no significant or even relevant effect

here. However, glia cells located in the hippocampus might lack expression of enzymes needed to

form allopregnanolone out of pregnenolone. If this was the case, allopregnanolone may exert a

negative impact on signal propagation in the hippocampus, but would not be increasingly

synthesized in the hippocampus following XBD administration. Consequently, XBD would not result

in inhibition of signal transduction in the hippocampus and thus not effect memory (assuming other

neurosteroids that positive allosteric modulate at GABAA receptors are not promoted either, which

could be the case, for XBD experiments in the amygdala did not result in suppression of neuronal

activity). XBD would still enhance pregnenolone synthesis and following other neurosteroids in the

hippocampus, but not allopregnanolone or any other positive allosteric GABAA receptor modulating

neurosteroids or XBD promotes diverse neurosteroids, which cancel out each others effects.

Supporting this theory, allopregnanolone has been observed to have a negative impact on memory

(Mayo et al. 1993, Zimmerberg et al. 1995). Further affirming this theory, enzymes for synthesis of

diverse neurosteroids have been determined to be region and neuron specific (Rupprecht et al.

2010). For instance, enzymes needed for formation of 5α-reduced neurosteroids have been found

to be almost absent in GABAergic hippocampal interneurons (Agís-Balboa et al. 2006).

4.1.3 Impact of diazepam, XBD and allopregnanolone on neuronal activity in the

thalamocortical connections

XBD impact on neuronal activity in the thalamocortical system was overall similar to diazepam

effect, with some distinct differences though. While XBD’s effect focused on merely diminishing the

intensity of intrathalamic and thalamocortical activity propagation, diazepam additionally affected

Discussion Result interpretation

44

the intensity in the intracortical experiment and the area of neuronal stimulus propagation in the

intrathalamic neuronal network. Ergo diazepam impact on the thalamocortical connections

exceeded XBD impact.

However, like diazepam, XBD clearly and significantly had an impact on intensity of signal

propagation in the thalamocortical connections arising from the ventrobasal nucleus of the

thalamus leading to the somatosensory cortex. Kita and his research group could not observe any

signs of sedative adverse effects upon XBD application to rodents (Kita et al. 2009). Furthermore,

subjects exposed to XBD intake did not report sedative effects (Rupprecht et al. 2009). In hindsight

it can be debated that a decline in the signal transduction in the thalamocortical connections might

be an additional reason for anxiety dissolution and not a cause for sedation. Hereby the train of

thought is that impairment of the thalamocortical connections might lead to a decrease in

awareness, but not on the physical condition of being awake. If this is the case, XBD might affect

alertness but would not sedate subjects. Encouraging this theory is the fact that the reticular

formation (as a part of the Reticular Activating System) is essentially involved in physical condition

states such as wakefulness and sleep-wake transitions (Trepel 2008) and might therefore be the

working point of the adverse effect of sedation upon diazepam application instead of the

thalamocortical system. To get more insight in this matter it would be interesting to perform clinical

studies investigating possible impact of XBD on awareness, concentration and reaction time. It also

would be interesting to see if XBD and diazepam show an effect in the Ascending Reticular

Activating System (RAS). Since up to date a slice preparation to properly inspect the RAS has not

been developed, there was no way to include this brain area in this dissertation.

The experiments with allopregnanolone in the thalamocortical system showed quite different

results. Allopregnanolone did not significantly affect the intrathalamic neuronal network. It did

however increase the area in the thalamocortical connections. Additionally, allopregnanolone

decreased the intensity of the stimulus propagation intracortical, however it did not in the

thalamocortical connections. The tendency of allopregnanolone to increase area in the

thalamocortical experiments might be considered as scattering of stimulus propagation, implying

that the once accurate and specific projection of excitation from the thalamus to a certain area of

the cortex is now affected and stimulus propagation left unguided.

The discrepancy in allopregnanolone and XBD results in the thalamocortical connections might

indicate that allopregnanolone is not the only neurosteroid promoted upon XBD application in the

thalamocortical connections. Since XBD rendered a quite different outcome, it is to be supposed

that other neurosteroids besides allopregnanolone conducted its actions, which might be due to a

different enzymatic equipment for NS synthesis in the thalamocortical system compared to the

hippocampus.

Discussion Clinical relevance, sources of error and outlook

45

For further insight into substance effect on the thalamocortical connections an analysis of alteration

of neuronal activity in each cortex layer upon thalamic stimulation was additionally undertaken.

While a few tendencies can be derived from this layer analysis (see chapter 4.1.3), due to

discrepancies in the results no ascertained conclusions can be drawn.

4.2 Clinical relevance, sources of error and outlook

While XBD and diazepam concentrations correspond with clinically used dosage, allopregnanolone

concentration was quite elevated in comparison to endogenous found concentrations. Little is

known about the XBD-induced synthesis of other neurosteroids than allopregnanolone. However,

the experiments with allopregnanolone served to determine general discrepancies to XBD. Even

though allopregnanolone has been applied at low doses, the effects of allopregnanolone on

neuronal activity might change remarkably when concentrations are adapted to physiological

levels.

Possible source of interference could have been the voltage sensitive dye di-4-ANEPPS, which is

known to have a modulating effect on GABA-receptors itself (Mennerick et al. 2010). However,

these effects can be considered negligible, since control recordings were equally affected as

recordings in the presence of substances.

Another TSPO ligand, alpidem, has already been approved for anxiety treatment in France in 1991

(Langer et al. 1990). Due to severe liver damage with profound and even lethal consequences it

was withdrawn in 1994 (Barki et al. 1993, Baty et al. 1994, Ausset et al. 1995). However, different

to XBD, alpidem also binds to GABAA receptors directly, which could also be the cause for liver

dysfunction (Langer et al. 1990). Nevertheless, further assessment of possible adverse effects and

tolerance in long-term clinical studies, including studies on diseased patients, are necessary to

investigate long-term tolerability and confirm clinical safety and relevance before XBD can be

introduced as a treatment option in anxiety disorders.

4.3 Conclusion

According to the data collected, XBD reduces neuronal activity in the amygdala, correspondingly

counteracting anxiety, while not significantly modulating neuronal networks in the hippocampus,

explaining the lack of anterograde amnesia. As for its alteration of neuronal activity in the

thalamocortical system, further investigations such as clinical studies determining XBD effect on

alertness need to be realized previous to final conclusions. To further elucidate the discrepancy

concerning the adverse sedative effect, the neurophysiological impact of diazepam and XBD on

Discussion Conclusion

46

brainstem areas involved in sleep-wake rhythms such as the reticular formation are of considerable

interest.

Similar to XBD, diazepam affects neuronal activity in the amygdala and the thalamocortical circuits,

but unlike XBD, also demonstrated diminishing effects on neuronal activity in the hippocampus,

which is in line with clinical observation of anterograde amnesia upon benzodiazepine application

(Rote-Liste 2013, 2015). In comparison, the presented data provides further evidence for XBD as

a promising anxiolytic drug to be superior to diazepam, regarding its side effect profile (Kita et al.

2009, Rupprecht et al. 2009).

The acquired data suggests that allopregnanolone is not the only neurosteroid formed upon XBD

application. This can be drawn from the fact that XBD and allopregnanolone utilization in the

thalamocortical connections resulted in different change of stimulation propagation. Furthermore,

in contrast to XBD, allopregnanolone reduced stimulation propagation in the hippocampus. This

indicates that, while allopregnanolone could affect neuronal networks in the amygdala, XBD

application does not lead to allopregnanolone formation in the hippocampus. This is in line with the

observation that neurosteroid biosynthesis is region and neuron specific and depends on TSPO

abundance and expression of the respective enzymes, responsible for genesis of the diverse

neurosteroids (Rupprecht et al. 2010). Region-specific synthesis of neurosteroids is subsequently

responsible for distinct modulation of neurotransmitter receptors in certain brain areas, resulting in

region-specific alteration of signal transduction. Additionally, distribution of GABAA receptor

subtypes adds to region specific alteration of signal transduction, since they vary in binding

capabilities (Hosie et al. 2006). Therefore, there are diverse parameters relevant for the

pharmacological profile of XBD, including TSPO abundance, distribution of the respective enzymes

needed for neurosteroid syntheses and GABAA receptor subtypes.

Abstract

47

5 Abstract

Despite the high prevalence of anxiety disorders in today’s society, suitable anxiolytic medication

needs yet to be established. Frequently used benzodiazepines show rapidly on-setting anxiolytic

effects. However, they elicit a broad range of adverse effects such as sedation and anterograde

amnesia. XBD, a ligand to the Translocator Protein 18 kDa (TSPO), is a promising new option.

XBD enhances TSPO mediated syntheses of neurosteroids, which have been shown to exert

anxiolytic effects. Clinical studies suggest XBD to lack unfavorable side effects commonly known

in benzodiazepine treatment. Like benzodiazepines, neurosteroids e.g. allopregnanolone act as

positive allosteric modulators at the GABAA receptor, though they occupy another binding site. The

discordance in the side effect profile is thought to origin in GABAA subtype distribution in the brain.

This present study investigates effects of XBD and diazepam on diverse brain areas using an

imaging technique, VSDI, that enables monitoring of signal propagation in the brain. The effects of

XBD and diazepam in diverse brain areas were monitored and compared. While both substances

showed considerable and similar impact on stimulus propagation in the amygdala, the anxiety

center of the brain, only diazepam affected neuronal activity in the hippocampus, the essential brain

area for memory consolidation. The collected data suggest similar effects on neuronal activity in

the amygdala (development of anxiety), while XBD lacks the negative effect of benzodiazepines

on neuronal activity in the hippocampus (known for memory formation). This is concordant with

clinical studies where XBD lacks the adverse effect of anterograde amnesia much known in

benzodiazepine treatment, while exerting similar anxiolytic properties. Like diazepam, XBD

affected neuronal activity in the thalamocortical system, which might be an indicator that XBD could

impair alertness and concentration. To investigate the mechanism of action of XBD, an additional

set of experiments was performed with allopregnanolone as presumably the most important

neurosteroid promoted upon XBD application. The discrepancies in neuronal activity observed after

XBD or allopregnanolone application in different brain regions may be explained by the fact that it

is highly probable that beside allopregnanolone, XBD enhances the syntheses of other

neurosteroids which are also pharmacological active. Furthermore, neurosteroid synthesis upon

TSPO activation depends on the specific enzymatic machinery needed for neurosteroid formation.

Which, at least in the hippocampus, does not allow the synthesis of allopregnanolone. Altogether,

the data collected provides further evidence that XBD promises to be superior to diazepam in

pharmacological treatment of anxiety disorders.

References

48

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Appendix

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7 Appendix

Used Substances

Sucrose-based-ACSF: pH ≈ 7,37 (saturated with carbogen) by Ying und Goldberg (Ying et al.

2005)

Substance Concentration (mM) Manufacturer

CaCl2 0,5 Sigma Aldrich

Glucose 11 Sigma Aldrich

Sucrose 234 Sigma Aldrich

KCl 2,5 Sigma Aldrich

MgSO4 10 Sigma Aldrich

NaHCO3 24 Sigma Aldrich

NaH2PO4 1,25 Sigma Aldrich

General ACSF: pH ≈ 7,37 (saturated with carbogen)

Substance Concentration (mM) Manufacturer

CaCl2 2 Sigma Aldrich

D(+)-Glucose 25 Sigma Aldrich

KCl 2,5 Sigma Aldrich

MgCl2 1 Sigma Aldrich

NaCl 125 Sigma Aldrich

NaHCO3 25 Sigma Aldrich

NaH2PO4 1,25 Sigma Aldrich

Appendix

61

Further Chemicals:

Substance Concentrations used Manufacturer

Allopregnanolone 1 µM (DMSO) Sigma Aldrich

Bicucullin 0,6 µM (DD) Sigma Aldrich

Di-4-Annepps see ch. 2.2.1 Sigma Aldrich

Diazepam 1 µM (DMSO) Sigma Aldrich

DMSO - Sigma Aldrich

Finasterid 10 µM (DMSO) Sigma Aldrich

Flumazenil 10 µM (DMSO) Sigma Aldrich

XBD 173 3 µM (70% Alcohol) Novartis

Appendix

62

Acknowledgements

Sincere thanks are given to everybody making this dissertation possible, especially:

My doctoral advisor, Prof. Dr. Gerhard Rammes for awarding me the topic and presenting me with

the opportunity to finalize my dissertation at the clinic of anesthesiology of the Klinikum Rechts der

Isar, to work in his charming research group, the excellent supervision as well as for reviewing my

thesis.

Dr. C. Mattusch for the splendid introduction to scientific working, outstanding mentoring and

helpfulness in all concerns.

As well as Martina Bürge, for her patients and utterly helpful guidance.

Dr. Matthias Eder, for letting me use the premises of his working group in the Max-Planck-Institute

for psychiatry as well as academic council.

The entire working group of AG Eder at the Max-Planck-Institute for the charming collaboration. To

be individually mentioned are Konrad Straub, Stefan Kratzer, Julien Dien, Jens Stephan and

Florian Hladky.

Last but certainly not least I want to thank Matthias Kreuzer for designing the Matlab-macros and

even more for his patience and encouraging words.